Compositions comprising decitabine and tetrahydrouridine and uses thereof

ABSTRACT

Embodiments of compositions and methods for the treatment of blood disorders and malignancies in a subject are described herein. In one embodiment, a composition for the treatment of a blood disorder or a malignancy in a subject comprises decitabine, tetrahydrouridine, and an excipient. In another embodiment, a method for the treatment of a blood disorder or a malignancy in a subject comprises the oral administration of a composition comprising decitabine and tetrahydrouridine. In some examples, the composition may be administered 1-3 times weekly.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims priority toU.S. patent application Ser. No. 15/875,727, filed Jan. 19, 2018, whichis a continuation of Ser. No. 15/044,805, filed Feb. 16, 2016, now U.S.Pat. No. 9,895,391, issued on Feb. 20, 2018, which is a continuation ofU.S. patent application Ser. No. 13/414,546, filed on Mar. 7, 2012, nowU.S. Pat. No. 9,265,785, issued on Feb. 23, 2016, which in turn claimspriority to U.S. Provisional Application No. 61/486,428, filed May 16,2011, and is a continuation-in-part of U.S. patent application Ser. No.13/141,669, now U.S. Pat. No. 9,259,469, issued Feb. 16, 2016, which wasfiled Aug. 5, 2011 as a national phase entry of InternationalApplication No. PCT/US2009/069035, filed Dec. 21, 2009, entitled“COMPOSITIONS COMPRISING DECITABINE AND TETRAHYDROURIDINE AND USESTHEREOF,” which in turn claims priority to U.S. Provisional ApplicationNos. 61/158,937, filed Mar. 10, 2009, and 61/139,710, filed Dec. 22,2008. The entire disclosures of these applications are herebyincorporated by reference in their entireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under Grant/Contract No.HL090513 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

TECHNICAL FIELD

Embodiments herein relate to the treatment of blood disorders andmalignancies. More specifically, disclosed herein are compositions andmethods for the treatment of blood disorders and malignancies in asubject, which methods comprise the oral administration of a compositioncomprising decitabine andtetrahydrouridine.

BACKGROUND

SCD is a serious congenital disease which affects 1 in 500African-Americans, as well as individuals of other racial backgrounds,exacting a substantial toll in morbidity and mortality upon theapproximately 100,000 Americans afflicted. SCD patients can havefrequent episodes of severe, debilitating pain, often requiringemergency room visits or hospitalization, and time off from work orschool. Although some patients respond to hydroxyurea (HU, the standardtreatment for symptomatic patients), and a few may be candidates forallogeneic stem cell transplantation, most patients continue to suffer.Furthermore, HU is used at doses that cause acute DNA damage andcytotoxicity, and has potential genotoxic, teratogenic andanti-fertility effects. Similarly, allogeneic stem cell transplantationis performed with cytotoxic conditioning and attendant risks oftreatment-related mortality.

A clinical research effort to develop pharmacologic inducers of HbFexpression culminated in FDA approval of the anti-metabolite HU to treatsymptomatic SCD in 1998. A recent follow-up of patients enrolled in thepivotal HU trial confirmed that a decreased risk of mortality in thesepatients correlates with HbF levels (Steinberg, M. H., et al. 2003 JAMA289: 1645-1651; Rosse, W. F., et al. 2000 Am Soc Hematol Educ Program2-17). However, HbF levels are not increased in approximately 40% of HUcompliant patients (Steiberg, M. H., et al. 1997 Blood 89: 1078-1088;Steinberg, M. H., et al. 1999 Expert Opin Investig Drugs 8:1823-1836;Atweh, G. F., et al. 2001 Curr Opin Hematol 8:123-130). Finally, HU isused at DNA-damaging, cytotoxic doses, potentially compounding the bonemarrow damage that accumulates in SCD.

Most cells in the body, including cancer cells or blood disorder cells,such as sickle cells, contain the same complement of genes. The functionand specialization of a cell is, therefore, determined by which of thesegenes are turned-on (activated), and which are turned-off (repressed).Activation refers to the expression of the protein encoded by the gene,while repression of the gene implies that the protein encoded by thatgene is expressed at lower levels or not at all. DNA methyl-transferase1 (DNMTI) is an enzyme which plays a critical and central role in themachinery that represses genes. Therefore, altering the levels of DNMTIwithin a cell can have powerful effects on the pattern ofgene-expression, function and specialization of a cell.

Decitabine (5-aza-2′-deoxycytidine) is a nucleoside analogue drug—a drugthat mimics a natural component of DNA. Decitabine is relatively uniqueamongst the large family of nucleoside analogue drugs in that it canirreversibly bind to and deplete DNMTI.

Cytidine deaminase (CAD) is an enzyme that is highly expressed in theliver and intestine and rapidly destroys decitabine within the body.Tetrahydrouridine (THU) is a safe and well-tolerated pyrimidinenucleoside analogue that inhibits cytidine deaminase. In humans, thecytidine deaminase gene is subject to non-synonymous single nucleotidepolymorphisms which produce variants of cytidine deaminase that havedifferences in enzymatic activity of 3-fold or more (Gilbert, J. A., etal. 2006 Clin Cancer Res 12, 1794-1803; Kirch, H. C., et al. 1998 ExpHematol 26, 421-425; Yue, L., et al. 2003 Pharmacogenetics 13, 29-38).

SUMMARY

Historically, decitabine was developed as an anti-metabolite or DNAdamaging drug intended to kill cancer cells by causing extensive damagewithin the cells. Its clinical or experimental application has not beenoptimized for the depletion of DNMTI. With an objective of usingdecitabine to deplete DNMTI to change cell behavior, antimetaboliteeffects that kill cells are undesirable. Optimization of decitabine todeplete DNMTI without causing other ‘off- target’ or toxic effects isdesired.

Optimizing decitabine to deplete DNMTI in vivo can have powerfultherapeutic benefits in a spectrum of diseases such as sickle celldisease, thalassemia, and cancers of multiple tissues. For example, insickle cell disease and β-thalassemia, by depleting DNMTI, decitabineprevents the repression of the fetal hemoglobin (HbF) gene. Theresulting increase in Hb abrogates the disease-causing effects of theabnormal sickle or thalassemia genes. Furthermore, the DNMTI depletionbydecitabine changes blood cell specialization, so that more red bloodcells are made, further addressing the debilitating anemia of theseconditions.

In cancer cells, DNMTI depletion by decitabine prevents the repressionof differentiation genes and renews the differentiation of the cancercells—the abnormal growth of the cancer cells is caused by a block intheir normal differentiation process, which is relieved by decitabine.Of especial note, DNMTI depletion in normal stem cells increases theirself-renewal; that is, DNMTI depletion increases the number of normalstem cells—the opposite of its effects on cancer cells. Therefore, DNMTIdepletion by decitabine could be an effective and very safe,well-tolerated cancer therapy.

Because of decitabine's unique ability to deplete DNMTI, and, therefore,alter the gene expression, function, and specialization of cells, andbecause DNMTI depletion by regimens of decitabine designed to depleteDNMTI without causing DNA damage increases HbF, and produces clinicalimprovement even in sickle cell disease patients with severe illnessdespite standard of care (Saunthararajah, et al. 2008 Brit J Haematol141(1): 126-9), decitabine is contemplated for treatment of these andother patients.

Because DNMTI depletion by decitabine induces the terminaldifferentiation and apoptosis of cancer cells, while increasing theself-renewal of normal stem cells (opposite effect on cancer cellsversus normal stem cells), decitabine is additionally contemplated fortreatment of these patients.

In one embodiment, the pharmacologic objective of therapy is to maximizea time-above-threshold concentration for depleting DNMTI (>0.1-0.2 μM),while avoiding high peak levels (>0.5-1 μM) that damage DNA. In anotherembodiment, the pharmacologic objective of therapy is to maximizetime-above-threshold concentration for depleting DNMTI (0.005-0.1 μM),while avoiding high peak levels (>0.5-1 μM) that damage DNA. In anotherembodiment, the pharmacologic objective of therapy is to maximize aduration of time within a desired concentration for depleting DNMTI(0.005-0.05 μM), while avoiding high peak levels (>0.5-1 μM) that damageDNA.

In another embodiment, the DNMTI -depleting effect should beintermittent. As a result, cells are allowed to divide and exhibit newbehaviors. Continuous exposure to decitabine may prevent cell divisionor even kill cells directly.

The currently known route of administration, regimens, and formulationsof decitabine produce high peak levels of the drug, which can kill cellsthrough anti-metabolite effects but produce very brieftime-above-threshold concentration for depleting DNMTI. Thus, thecurrently known route of administration, regimens, and formulations ofdecitabine do not deplete DNMTI intermittently to allow cell division,but, rather, produce cytotoxic or cytostatic effects. The destruction ofdecitabine by the enzyme cytidine deaminase (CDA) produces anabbreviated half-life in vivo of <20 minutes (despite an in vitrohalf-life of 5-9 hours) (Liu, Z., et al. 2006 Rapid Common Mass Spectrum20: 1117-1126). This drastic reduction in half-life is a significantbarrier to effective in vivo translation of in vitro observations.Pharmacogenomic variation in CDA (Gilbert, J. A., et al. 2006 ClinCancer Res 12, 1794-1803; Kirch, H. C., et al. 1998 Exp Hematol 26,421-425; Yue, L., et al. 2003 Pharmacogenetics 13, 29-38) produces largeinter-individual variation in pharmacokinetics (PK) and clinicaleffects. Currently, injections or infusions of decitabine must beadministered in the clinic or hospital, severely limiting its use insickle cell disease, where the goal is chronic disease modification forthe lifetime of the patient. In addition, intestinal CDA-mediateddestruction severely limits its oral bioavailability; while the in vitrohalf-life of decitabine is 5-9 hrs, it has an abbreviated half-life of<20 minutes in vivo because of CDA-mediated destruction) (Liu, Z., etal. 2006 Rapid Commun Mass Spectrom 20:1117-1126; Liu, Z., et al. 2007Nucleic Acids Res 35:e31), impeding the proposed treatment paradigm ofmulti-year, chronic therapy to produce sustained life-long therapeuticbenefits. Malignant cells can develop resistance by destroyingdecitabine with CDA (Ohta, T., et al. 2004 Oncol Rep 12:1115-1120;Hubeek, I., et al. 2005 Br J Cancer 93:1388-1394; Huang, Y., et al. 2004Cancer Res 64:4294-4301), and may find sanctuary from decitabinetherapeutic effects by residing in tissues with high levels of CDA.

For at least the above reasons, an oral route of administration ofdecitabine is contemplated herein. Such oral administration isconsidered herein to decrease peak levels and increase thetime-above-threshold concentration for depleting DNMTI, to enablechronic, frequent but not daily (i.e., metronomic) therapy to sustainlife-long therapeutic effects while allowing cell division andminimizing toxicity, and to enable wide-spread use of the drug acrossthe globe. Additionally contemplated herein to address the limitationsand issues iterated above is the combination of decitabine withtetrahydrouridine (THU) for oral administration.

THU inhibits CDA, exhibits a benign toxicity profile and awell-characterized PK, overcomes the intestinal and liver first-passbarriers to oral bioavailability of decitabine, and addressespharmacogenomic variation in CDA, which produces large inter- individualvariation in decitabine PK and therapeutic effects. THU can produce amore predictable effect of a decitabine dose from individual toindividual, increase the time-above-threshold concentration ofdecitabine for depleting DNMTI, remove sanctuary sites for malignantcells from decitabine therapeutic effects, and directly address onemechanism of cancer cell resistance to the therapeutic effects ofdecitabine.

Accordingly, in one embodiment, a composition fororal administrationcomprises decitabine and THU. In one example, the composition for oraladministration comprises about 10.0 to about 150 mg/m² decitabine andabout 100 to about 500 mg/m² THU. In another example, the compositionfor oral administration comprises about 0.01 to about 9.9mg/m²decitabine and about 100 to about 500 mg/m² THU. In anotherexample, the composition for oral administration comprises about 0.1 toabout 9.9 mg/m²decitabine and about 300 to about 600 mg/m² THU. Inanother example, the composition for oral administration comprises about0.035 to about 5.9 mg/m² decitabine and about 300 to about 600 mg/m²THU. In another example, the composition for oral administrationcomprises about 1.25 to about 4.9 mg/m² decitabine and about 350 toabout 450 mg/m² THU. In another example, the composition for oraladministration comprises about 5.0 to about 9.9 mg/m²decitabine andabout 350 to about 450 mg/m² THU. In yet another example, thecomposition for oral administration comprises about 3.0 to about 7.0mg/m²decitabine and about 400 mg/m² THU. In another example, thecomposition for oral administration comprises about 4.0 to about 6.0mg/m²decitabine and about 400 mg/m² THU. For example, the compositionmay comprise about 5.0 mg/m²decitabine and about 400 mg/m² THU.

In a particular embodiment, a composition for oral administrationcomprises about 1.25 to about 7.0 mg/m² decitabine and about 350 toabout 450 mg/m² THU, and may be formulated and/or administered such thatthe THU is bioavailable for some length of time, such as 15-180 minutes,before the decitabine is bioavailable. Unexpectedly, as describedfurther below, such compositions may provide improved concentration-timeprofiles for DNMT1 depletion with reduced cytotoxicity, resulting insafer and more efficacious DNMT1-targeted therapy. Surprisingly, innon-human primates, such compositions were found to provide a sustainedincrease in Hbf, and a larger cumulative increase in HbF, than otherwisesimilar compositions comprising 10 mg/m²decitabine (both administeredwith 400 mg/m²THU, bioavailable 60 min before decitabine). This resultindicates that a composition for oral administration comprising about1.25 to about 7.0 mg/m² decitabine and about 350 to about 450 mg/m² THU,made bioavailable for some time before the decitabine, may offerunexpected advantages in the treatment of blood disorders andmalignancies as described herein. Likewise, such compositions for oraladministration may provide an extended time above minimum decitabineconcentrations required for S-phase specific depletion of DNMT1, whileavoiding undesirable higher peak concentrations that cause DNA damageand cytotoxicity. Such compositions for oral administration may beadministered to a subject 1, 2, or 3 days a week on non-consecutive daysover multiple weeks to provide a safer, more efficacious, and moreconvenient method of treatment.

Compositions for oral administration may be formulated in variousdosages to treat subjects of varying sizes/weight. For example, thecomposition for oral administration can comprise 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 mg/m²decitabine and 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 mg/m²THU.

In another embodiment, the composition is for treating a blood disorderin a subject, such as a human subject. In yet another embodiment, theblood disorder is a hemoglobinopathy or a thalassemia. For example, thehemoglobinopathy may be sickle cell disease.

In another embodiment, the composition is for treating hematological orsolid malignancies, such as cancers affecting the blood, bone marrow,and lymph nodes, including leukemia, lymphoma, and multiple myeloma, aswell as related disorders like myelodysplastic syndrome,myeloproliferative disease, myelofibrosis, amyloid disorders, anemiaassociated with malignancy, anemia associated with inflammationincluding rheumatoid arthritis and inflammatory bowel diseases, anemiaof chronic renal failure, anemia associated with chronic infections suchas HIV or hepatitis, anemia due to thrombocytopenia associated withmalignancy, idiopathic thrombocytopenia purpura and viral diseasesincluding virally-related malignancies. Examples of virally-relatedmalignancies include EBV malignancies, including, without limitation,Burkitt's lymphoma, lymphomas associated with immunosuppression, othernon-Hodgkin's lymphomas, Hodgkin's disease, nasopharyngeal carcinoma,gastric adenocarcinoma, lymphoepithelioma-like carcinomas, andimmunodeficiency-related leiomyosarcoma.

In another embodiment, the composition is for treating cancers thataffect other tissues, such as cancers affecting the brain, head, neck,thyroid, bones, muscle, lung, esophagus, stomach, intestine, breasts,prostate, testes, ovaries, uterus, vagina, and skin.

In another embodiment, a composition for oral administration comprisesTHU and decitabine combined in a single capsule or tablet. In someexamples, a composition for oral administration may comprise about 100to 1000 mg THU and about 1 to 200 mg decitabine. Some compositions fororal administration may comprise about 400 to 1000 mg THU and about 1 to15 mg decitabine. Other compositions for oral administration maycomprise 15 to 200 mg decitabine and 400 to 1000 mg THU. Still othercompositions for oral administration may comprise about 5 to 10 mgdecitabine and 600 to 700 mg THU. In one example, a composition in theform of a capsule is contemplated comprising about 500 mg THU and about100 mg decitabine. In another example, a composition in the form of acapsule is contemplated comprising about 650 mg THU and about 8 mgdecitabine.

In some embodiments, a composition for oral administration maybe used toprovide an oral regimen approximating 500 mg/m² THU combined with 10 to100 mg/m²decitabine to produce a plasma concentration of decitabine of0.1-0.5 μM in a subject, such as a human subject. In other embodiments,a composition for oral administration may be used to provide an oralregimen approximating 400 mg/m² THU combined with 1.5 to 7.0mg/m²decitabine to produce a plasma concentration of decitabine of0.005-0.05 μM in a subject, such as a human subject. In otherembodiments, a composition for oral administration may be used toprovide an oral regimen approximating 400 mg/m² THU combined with 4.0 to6.0 mg/m²decitabine to produce a plasma concentration of decitabine of0.005-0.05 μM in a subject, such as a human subject. In otherembodiments, a composition for oral administration may be used toprovide an oral regimen approximating 400 mg/m² THU combined with 0.5 to9.9 mg/m²decitabine to produce a plasma concentration of decitabine of0.005-0.1 μM in a subject, such as a human subject.

In yet another embodiment, a composition of the disclosure isadministered once a week or once every two weeks to patients sufferingfrom a blood disorder. In still another embodiment, a composition of thedisclosure is administered between once to three times per week topatients with cancer. In yet another embodiment, a composition of thedisclosure is administered between once every two weeks to as often asthree times per week in patients at risk of developing hematological orsolid malignancy, or at risk of having a relapse in a previous diagnosisof hematological or solid malignancy.

In yet another example, the composition for oral administration is inthe form of a tablet, pill, capsule, lozenge, or other solid oralformulation that comprises about 0.25 to about 15 mg decitabine andabout 500 to 1000 mg THU. In other examples, the composition for oraladministration comprises about 0.5 to about 9.9 mg/m²decitabine andabout 350 to about 450 mg/m² THU. A composition for oral administrationmay comprise, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3,10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5,11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7,12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9,14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1,15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3,16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5,17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7,18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9,or 20.0 mg decitabine and 300, 325, 350, 375, 400, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850,875, 900, 925, 950, 975, or 1000 mg THU.

In another embodiment, a composition for oral administration is providedin the form of a capsule, comprising THU and decitabine, wherein the THUis released more quickly than the decitabine. For example, the THU mightbe subject to a faster dissolution rate, or the THU might be located atthe surface of the capsule, while the decitabine is located inside thecapsule. In one embodiment of such a composition, the THU isbio-available about 15 to about 180 minutes before the decitabine, inanother, about 30 to about 90 minutes before the decitabine, in another,about 60 minutes before the decitabine. The THU may also be administeredseparately, in succession (with THU first, then decitabine).

In another embodiment, the composition is stored with adessicant. Thiscould serve to extend the shelf-life of the composition and facilitateits distribution and use on a global scale.

In another embodiment, the disclosure provides a method for treating ablood disorder in a subject comprising administration of a compositioncomprising decitabine and tetrahydrouridine as described above. In yetanother embodiment, the disclosure provides a method for treating acancer in a subject comprising administration of a compositioncomprising decitabine and tetrahydrouridine as described above. In afurther embodiment, the administration occurs 1 -3 times per week. Forexample, a composition comprising decitabine and tetrahydrouridine maybe administered orally to a subject twice per week or three times perweek.

In one aspect, the disclosure provides a composition for oraladministration comprising about 10 to about 150 mg/m²decitabine andabout 100 to about 500 mg/m²tetrahydrouridine and a pharmaceuticallyacceptable excipient. In another aspect, the disclosure provides acomposition for oral administration comprising about 1 to about 9.9mg/m² decitabine and about 100 to about 500 mg/m² tetrahydrouridine anda pharmaceutically acceptable excipient. In another aspect, thedisclosure provides a composition for oral administration comprisingabout 100 mg decitabine and about 500 mg tetrahydrouridine and apharmaceutically acceptable excipient. In another aspect, the disclosureprovides a composition for oral administration comprising about 8 mgdecitabine and about 650 mg tetrahydrouridine and a pharmaceuticallyacceptable excipient.

In one embodiment, a composition of the disclosure is for treating ablood disorder in a subject. In yet another embodiment, the blooddisorder is a hemoglobinopathy or a thalassemia. In additionalembodiments, the hemoglobinopathy is a sickle cell disease, and thethalassemia is a beta thalassemia (for example, hemoglobin E betathalassemia).

In another embodiment, a composition of the disclosure is for treating ahematological or solid malignancy in a subject. In yet anotherembodiment, the malignancy is selected from the group consisting ofleukemia, lymphoma, multiple myeloma, cancer of the brain, cancer of thehead, cancer of the neck, cancer of the mouth, cancer of the pharynx,cancer of the esophagus, cancer of the stomach, cancer of the intestine,cancer of the thyroid, cancer of the lungs, cancer of the mediastinum,cancer of the thymus, cancer of the mesothelium, cancer of theperitoneum, cancer of the bone, cancer of the muscle, cancer of theskin, cancer of the prostate, cancer of the breasts, cancer of theovaries, cancer of the uterus, cancer of the vagina, and virally relatedmalignancy. In an additional embodiment, the virally-related malignancyis an EBV malignancy.

In another embodiment of a composition, the tetrahydrouridine isbio-available about 15 to about 180 minutes before the decitabine. Inanother embodiment, the tetrahydrouridine is bio-available about 30 toabout 60 minutes before the decitabine. In one embodiment, the THU anddecitabine are administered concurrently. In another embodiment, the THUis administered first, and the decitabine is administered later.

In another aspect, the disclosure provides a composition for oraladministration in the form of a capsule or tablet comprising decitabineand tetrahydrouridine and a pharmaceutically acceptable excipient,wherein the tetrahydrouridine is bio-available about 15 to about 180minutes before the decitabine; or about 30 to about 60 minutes beforethe decitabine. In one embodiment, the tetrahydrouridine is located atthe surface of the capsule or tablet, and the decitabine is locatedwithin the capsule or tablet. In yet another embodiment of thecomposition of the disclosure, the THU is administered first in acapsule or tablet, and the decitabine is administered later in a secondcapsule or tablet.

In another aspect, the disclosure provides a method for treating a blooddisorder in a subject, comprising administering to the subject acomposition as described herein. In one embodiment, the blood disorderis a hemoglobinopathy or a thalassemia. In another embodiment, thesubject is provided an additional form of therapy.

In another aspect, the disclosure provides a method for treating ahematological or solid malignancy in a subject, comprising administeringto the subject a composition as described herein. In one embodiment, themalignancy is selected from the group consisting of leukemia, lymphoma,multiple myeloma, cancer of the brain, cancer of the head, cancer of theneck, cancer of the mouth, cancer of the pharynx, cancer of theesophagus, cancer of the stomach, cancer of the intestine, cancer of thethyroid, cancer of the lungs, cancer of the mediastinum, cancer of thethymus, cancer of the mesothelium, cancer of the peritoneum, cancer ofthe bone, cancer of the muscle, cancer of the skin, cancer of theprostate, cancer of the breasts, cancer of the ovaries, cancer of theuterus, cancer of the vagina, and virally related malignancy. In anotherembodiment, the virally related malignancy is an EBV malignancy. In yetanother embodiment, the subject is provided an additional form oftherapy.

In another aspect, the disclosure provides a method for decreasing theinter-individual variation in decitabine pharmacokinetics and/orclinical effects in subjects, comprising administering to the subjects acomposition as described herein.

In still another aspect, the disclosure provides a method forextendingthe time-above-threshold concentration for depleting DNMTI withdecitabine in a subject and avoiding DNA-damaging high peak levels ofdecitabine, comprising administering to the subject a composition asdescribed herein.

In one embodiment of a method, the subject is human. In anotherembodiment, the method further comprises obtaining the composition.

Other aspects are described in or are obvious from the followingdisclosure and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of Examples, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying figures, in which:

FIG. 1 A quantifies, in bar graph form, DNA damage assessed in normalCD34+ hematopoietic cells subject to various decitabine concentrations.FIG. 1B shows histograms depicting DNA damage assessed byphosphorylation of histone H2AX. FIG. 1C graphically depicts S-phase/G2Mwith various concentrations of decitabine treatment.

FIG. 2A graphically depicts cell counts for CD34+ cells, AMLI-ETO CD34+cells, and KASUMI-I cells exposed to Ara-C vs. two different doses ofdecitabine. FIG. 2B shows, in bar graph form, colony-forming ability ofnormal CD34+ cells and AMLi-ETO CD34+ cells treated with decitabine vs.Ara-Cover time.

FIG. 3A shows a table providing FAB type and cytogenic abnormalities forsamples from various patients. FIG. 3B graphically depicts the cellcounts at day 7 in untreated vs. decitabine-treated cells.

FIG. 4 graphically depicts cell counts in untreated vs.decitabine-treated cells at day 9.

FIG. 5A graphically depicts tumor volume over time in mice treated withPBS (control) vs. decitabine vs. Sunitinib vs. a combination ofdecitabine and Sunitinib. FIG. 5B graphically depicts cell number overtime for the mice.

FIG. 6 shows, in bar graph form, the repression of HoxB4, Bmi-1, andcKIT and the activation of Mcsfr, Gmcsfr, and F4/80 over time in PUERcells.

FIG. 7A graphically depicts cell proliferation over time of treatmentwith OHT vs. OHT+decitabine vs. OHT followed by decitabine. FIG. 7Bgraphically depicts macrophage differentiation (F4/80) and stem cells(c-KIT) over time of treatment with OHT vs. OHT+decitabine vs. OHTfollowed by decitabine. FIG. 7C shows, in bar graph form, Bmil, HoxB4,cKIT , F4/80, Gmcsfr, and Mcsfr over time of treatment with OHT vs.OHT+decitabine vs. OHT followed by decitabine.

FIG. 8 shows, in bar graph form, Bmil, HoxB4, cKIT, F4/80, Gmcsfr, andMcsfr over time in PUERshRunx1 cells.

FIG. 9A shows, in bar graph form, PU.1 and CEBPα. FIG. 9B shows, in bargraph form, CEB Pϵ and GATA-I in various patient samples. FIG. 9C lists,in table form, the WHO classification and cytogenic abnormalities foreach of the samples. FIG. 9D shows, in bar graph form, precursor genemethylation and differentiation gene methylation for normal CD34+ cells,normal bone marrow cells, MDS cells, and AML cells.

FIG. 10 plots the survival of leukemic mice untreated vs. treated withdecitabine.

FIG. 11 graphically depicts decitabine peak levels andtime-above-threshold for DNMTI depletion produced by subcutaneous vs.oral decitabine.

FIGS. 12A and 12B show, in bar graph form, DNMTI depletion indecitabine-sensitive and decitabine-resistant cell lines (in terms of %control growth for the bar graphs).

FIG. 13 graphically depicts HbF expression elevation over timeassociated with decitabine.

FIG. 14 graphically depicts plasma concentration-time curves ofdecitabine in non-human primates.

FIG. 15 graphically depicts plasma concentration-time curves ofdecitabine following oral administration to baboons at 10 mg/kg.

FIG. 16 graphically depicts plasma concentration-time curves ofdecitabine in baboons following oral administration alone or 60 minutesafter THU.

FIG. 17 graphically depicts plasma concentration-time curves ofdecitabine in baboons following oral administration alone at 5 mg/kg or60 minutes after 2 or 20 mg/kg THU.

FIG. 18A depicts, in bar graph form, the distribution of AUC in 7animals treated with decitabine 10 mg/kg alone by oral gavage or THU 20mg/kg by oral gavage followed by decitabine 5 mg/kg by oral gavage 60minutes later. Horizontal line in box−plot=median, boxboundaries=interquartile range, connecting diagonal line joins the meanin the two groups. The wide separation of median and mean in thedecitabine only group is narrowed substantially in the decitabine-THUgroup. The difference in medians between the two groups was notstatistically significant (p=0.22,Wilcoxon). The difference in meansbetween the two groups was not statistically significant (p=0.08, pairedt-test). FIG. 18B depicts, in bar graph form, the results of A brokendown by individual animals.

FIGS. 19A-19E illustrates effects of DAC on DNMT1 depletion, DNA damage,and apoptosis in normal hematopoietic precursors. FIG. 19A: DAC 0.005 μMdepletes DNMT1 in normal hematopoietic precursors. FIG. 19B-C: DAC>0.5μM was required to induce measurable DNA damage. FIG. 19D: DAC>0.5 μMwas required to induce apoptosis. FIG. 19E: DAC up to 0.5 μM incombination with THU did not cause significant DNA damage.

FIGS. 20A-20D illustrates plasma concentration-time curves followingintravenous (IV) decitabine (DAC) 10 mg/m2 (0.5 mg/kg), subcutaneous(SC) DAC 10 mg/m2 (0.5 mg/kg) and oral gavage (Oral) DAC 200 mg/m² (10mg/kg) administration to baboons. FIG. 20A shows IV and oraladministration in PA7472. FIG. 20B shows IV and oral administration inPA7482. FIG. 20C shows SC and oral administration in PA7254. FIG. 20Dshows SC and oral administration in PA7258.

FIGS. 21A-21B illustrates identification of dose and timing of oral THUto increase oral bioavailability of DAC in nonhuman primates. In 2female baboons, PA7470 and PA7484 (selected for high and lowbioavailability of oral DAC alone, respectively), THU 400 mg/m² (20mg/kg) 60 minutes before DAC 100 mg/m² (5 mg/kg) produced higher DACconcentrations than THU 40 mg/m² (2 mg/kg; FIG. 21A-B). In these samebaboons after a washout period, THU 400 mg/m² 60 minutes before DACproduced higher DAC concentrations than THU 400 mg/m² administeredsimultaneously or 30 minutes before DAC (FIG. 21A-B).

FIGS. 22A-22C illustrates effects of prior administration of oral THU onoral bioavailability and interindividual variability in pharmacokineticsof DAC. FIG. 22A shows DAC concentration-time profiles in 8 baboonsadministered oral DAC 200 mg/m2 (10 mg/kg). FIG. 22B shows DACconcentration-time profiles in the same 8 baboons administered DAC athalf the dose (100 mg/m2 [5 mg/kg]) 60 minutes after THU 400 mg/m2 (20mg/kg; THU-DAC). FIG. 22C shows AUClast in 7 animals administered DACalone versus the same 7 animals receiving DAC at half the dose afterTHU.

FIG. 23 illustrates the DAC concentration-time profile in miceadministered DAC alone or DAC 60 minutes after THU. Drugs wereadministered by oral gavage. Dots show values from 3 mice for each timepoint in each treatment group. THU-DAC indicates DAC 0.4 mg/kg 60minutes after THU 167 mg/kg. DAC indicates DAC 0.4 mg/kg 60 minutesafter vehicle.

FIGS. 24A-24E illustrates pharmacodynamic effects of repeat dose oralTHU- decitabine in non-human primates. A baboon with relatively low anda baboon with relatively high oral THUDAC bioavailability in thepharmacokinetic (PK) studies (baboon numbers PA7472 and PA7470,respectively) received DAC 5 mg/m2, and another pair from each end ofthe PK range (baboon numbers PA7482 and PA7484, respectively) receivedDAC 10 mg/m2. DAC was administered 60 minutes after THU 400 mg/m2 2×/wkfor 8 weeks. FIG. 24A shows platelet counts during drug administration.FIG. 24B shows absolute neutrophil counts during drug administration.FIG. 24C shows phospho-H2AX (γH2AX) labeling of BM cells 96 hours afterTHU-DAC administration in week 8 in baboon number PA7472. FIG. 24D showsHbF expression during treatment. FIG. 24E shows decrease in methylationof developmentally responsive CpG in the γ-globin gene (HBG) promoterafter drug administration in baboon numbers PA7472 and PA7484.

FIGS. 25A-25F illustrates pharmacodynamic effects of repeat dose oralTHU- decitabine in non-human primates. Decitabine 5 mg/m2 (PA7484) or2.5 mg/m2 (PA7472) 60 minutes after THU 400 mg/m2 3×/week wasadministered for 5 weeks to PA7484 and for 3 weeks to PA7472. FIG. 25Ashows platelet counts in PA7484. FIG. 25B shows absolute neutrophilcounts (ANC) in PA7484. FIG. 25C shows HbF % in PA7484. FIG. 25D showsplatelet counts in PA7472. FIG. 25E shows ANC in PA7472. FIG. 25F showsHbF % in PA7472.

DETAILED DESCRIPTION I. Definitions

The term “treating”, as used herein, refers to altering the diseasecourse of the subject being treated. Therapeutic effects of treatmentinclude, without limitation, preventing occurrence or recurrence ofdisease, alleviation of symptom(s), diminishment of direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

Hematological malignancies are a group of neoplasms that arise throughmalignant transformation of bone marrow derived cells. They can besubdivided into myeloid and lymphoid disorders and include, withoutlimitation, acute lymphoblastic leukemia, chronic lymphoid leukemia,diffuse large B-cell lymphoma, follicular centre lymphoma, Hodgkinslymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom'smacroglobulinemia, myeloma, monoclonal gammopathy of uncertainsignificance, large granular lymphocyte syndrome, T-prolymphocyticsyndrome, Sezary syndrome, lymphoma, angio-immunoblastic lymphoma,anaplastic large cell lymphoma, mycosis fungoides, lymphomatoidpapulosis, small intestinal lymphoma, acute myeloid leukemia,myelodysplastic syndrome, myeloproliferative disorders, myelofibrosis,paroxysmal nocturnal hemoglobinuria, aplastic anemia, anemia associatedwith malignancy, thrombocytopenia associated with malignancy,virally-related malignancies, post-transplant lymphoproliferativesyndrome, NK/T lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, andnon-Burkitt's small cell lymphoma. Chronic lymphocytic leukemia,non-Hodgkin lymphoma, and myeloid leukemia are particularly prevalentmalignancies contemplated for treatment herein.

Solid tumor malignancies are a group of neoplasms that arise through themalignant transformation of cells in non-blood-related tissues. Theseinclude, without limitation, malignancies of the brain, head, neck,mouth, pharynx, esophagus, stomach, intestine, thyroid, lungs,mediastinum, thymus, mesothelium, peritoneum, bone, muscle, skin,prostate, breasts, ovaries, uterus, and vagina.

Hemoglobinopathies and thalassemias can both be characterized as “blooddisorders” and are caused by abnormalities in the globin genes. Blooddisorders include disorders that can be treated, prevented, or otherwiseameliorated by the administration of a compound of the disclosure. Ablood disorder is any disorder of the blood and blood-forming organs.The term blood disorder includes nutritional anemias (e.g., irondeficiency anemia, sideropenic dysphasia, Plummer-Vinson syndrome,vitamin B 12 deficiency anemia, vitamin B 12 deficiency anemia due tointrinsic factor, pernicious anemia, folate deficiency anemia, and othernutritional anemias), myelodysplastic syndrome, bone marrow failure oranemia resulting from chemotherapy, radiation or other agents ortherapies, hemolytic anemias (e.g., anemia due to enzyme disorders,anemia due to phosphate dehydrogenase (G6PD) deficiency, favism, anemiadue to disorders of glutathione metabolism, anemia due to disorders ofglycolytic enzymes, anemias due to disorders of nucleotide metabolismand anemias due to unspecified enzyme disorder), thalassemia,α-thalassemia, β-thalassemia (for example, hemoglobin E betathalassemia), δβ-thalassemia, thalassemia trait, hereditary persistenceof fetal hemoglobin (HPFP), and other thalassemias, sickle celldisorders (sickle cell anemia with crisis, sickle cell anemia withoutcrisis, double heterozygous sickling disorders, sickle cell trait andother sickle cell disorders), hereditary hemolytic anemias (hereditaryspherocytosis, hereditary elliptocytosis, other hemoglobinopathies andother specified hereditary hemolytic anemias, such as stomatocyclosis),acquired hemolytic anemia (e.g., drug-induced autoimmune hemolyticanemia, other autoimmune hemolytic anemias, such as warm autoimmunehemolytic anemia, drug-induced non-autoimmune hemolytic anemia,hemolytic-uremic syndrome, and other non-autoimmune hemolytic anemias,such as microangiopathic hemolytic anemia); aplastic anemias (e.g.,acquired pure red cell aplasia (erythoblastopenia), other aplasticanemias, such as constitutional aplastic anemia and fanconi anemia,acute post-hemorrhagic anemic, and anemias in chronic diseases),coagulation defects (e.g., disseminated intravascular coagulation(difibrination syndrome)), hereditary factor VIII deficiency (hemophiliaA), hereditary factor IX deficiency (Christmas disease), and othercoagulation defects such as Von Willebrand's disease, hereditary factorXi deficiency (hemophilia C), purpura (e.g., qualitative plateletdefects and Glanzmann's disease), neutropenia, agranulocytosis,functional disorders of polymorphonuclear neutrophils, other disordersof white blood cells (e.g., eosinophilia, leukocytosis, lymophocytosis,lymphopenia, monocytosis, and plasmacyclosis), diseases of the spleen,methemoglobinemia, other diseases of blood and blood forming organs(e.g., familial erythrocytosis, secondary polycythemia, essentialthrombocytosis and basophilia), thrombocytopenia, infectious anemia,hypoproliferative or hypoplastic anemias, hemoglobin C, D and E disease,hemoglobin lepore disease, and HbH and HbS diseases, anemias due toblood loss, radiation therapy or chemotherapy, or thrombocytopenias andneutropenias due to radiation therapy or chemotherapy, sideroblasticanemias, myelophthisic anemias, antibody-mediated anemias, and certaindiseases involving lymphoreticular tissue and reticulohistiocytic system(e.g., Langerhans' cell hystiocytosis, eosinophilic granuloma,Hand-Schuller-Christian disease, hemophagocytic lymphohistiocytosis, andinfection-associated hemophagocytic syndrome).

The thalassemias are classified according to which chain of thehemoglobin molecule is affected. In α thalassemias, production of the αglobin chain is affected, while in β thalassemia, production of the βglobin chain is affected, β globin chains are encoded by a single geneon chromosome 11. Beta thalassemias are due to mutations in the HBB geneon chromosome 11.

The severity of the disease depends on the nature of the mutation.Mutations are characterized as (β° or β thalassemia major) if theyprevent any formation of β chains (which is the most severe form of betathalassemia); they are characterized as (β⁺ or β thalassemia intermedia)if they allow some β chain formation to occur. In either case, there isa relative excess of α chains, but these do not form tetramers: rather,they bind to the red blood cell membranes, producing membrane damage,and at high concentrations they form toxic aggregates.

The term “pharmaceutically acceptable excipient”, as used herein, refersto carriers and vehicles that are compatible with the active ingredient(for example, a compound of the disclosure) of a pharmaceuticalcomposition of the disclosure (and preferably capable of stabilizing it)and not deleterious to the subject to be treated. For example,solubilizing agents that form specific, more soluble complexes with thecompounds of the disclosure can be utilized as pharmaceutical excipientsfor delivery of the compounds. Suitable carriers and vehicles are knownto those of extraordinary skill in the art. The term “excipient” as usedherein will encompass all such carriers, adjuvants, diluents, solvents,or other inactive additives. Suitable pharmaceutically acceptableexcipients include, but are not limited to, water, salt solutions,alcohol, vegetable oils, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid monoglycerides and diglycerides, petroethralfatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc.The pharmaceutical compositions of the disclosure can also be sterilizedand, if desired, mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like, which do not deleteriously react withthe active compounds of the disclosure.

The term “bio-available”, as referred to herein, refers to when theactive agent (tetrahydrouridine or decitabine) can be absorbed and usedby the body. “Orally bio-available” indicates that the agent has beentaken by mouth and can be absorbed and used by the body.

The term “subject” as used herein refers to a vertebrate, preferably amammal, more preferably a primate, still more preferably a human.Mammals include, without limitation, humans, primates, wild animals,feral animals, farm animals, sports animals, and pets.

The term “obtaining” as in “obtaining the composition” is intended toinclude purchasing, synthesizing, or otherwise acquiring the composition(or agent(s) of the composition).

The terms “comprises”, “comprising”, are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

The disclosure can be understood more fully by reference to thefollowing detailed description and illustrative examples, which areintended to exemplify non-limiting embodiments of the disclosure.

II Additional Embodiments

Pharmaceutical Compositions

In one embodiment, pharmaceutical compositions and dosage forms compriseabout 10 to about 150 mg/m² decitabine and about 100 to about 500 mg/m²THU and a pharmaceutically acceptable excipient, in relative amounts andformulated in such a way that a given pharmaceutical composition ordosage form reactivates fetal hemoglobin (HbF) expression and/or expandsnormal hematopoietic stem cells and/or causes a shift to theerythropoietic lineage. In another embodiment, pharmaceuticalcompositions and dosage forms comprise about 1.0 to about 9.9 mg/m²decitabine and about 100 to about 500 mg/m² THU and a pharmaceuticallyacceptable excipient, in relative amounts and formulated in such a waythat a given pharmaceutical composition or dosage form reactivates fetalhemoglobin (HbF) expression and/or expands normal hematopoietic stemcells and/or causes a shift to the erythropoietic lineage. In anotherembodiment, the pharmaceutical compositions and dosage forms compriseabout 100 mg decitabine and about 500 mg THU and a pharmaceuticallyacceptable excipient. In another embodiment, the pharmaceuticalcompositions and dosage forms comprise about 5 to10 mg decitabine andabout 400 to 800 mg THU and a pharmaceutically acceptable excipient.

In another embodiment, pharmaceutical compositions anddosage formscomprise about 0.027 to 0.27 mg/kg decitabine and about 8 to about 14mg/kg THU and a pharmaceutically acceptable excipient, in relativeamounts and formulated in such a way that a given pharmaceuticalcomposition or dosage producing in the subject a peak decitabine plasmaconcentration of <0.5 μM, with the decitabine plasma concentrationmaintained at or above 0.005 μM for at least 60 minutes. In someembodiments, the given pharmaceutical composition or dosage may producein the subject a peak decitabine plasma concentration of <0.5 μM andmaintain the decitabine plasma concentration at 0.005 μM to 0.1 μM forat least 60 minutes.

In another embodiment, the compositions are formulated in such a waythat a given pharmaceutical composition or dosage form decreases theaberrant repression of differentiation-related genes in hematological orsolid malignancies, thus reducing or inhibiting the growth oftransformed (cancer) cells. In another embodiment, such pharmaceuticalcompositions and dosage forms comprise one or more additional activeagents. For the treatment of hematological or solid malignancies, suchadditional active agents include chemotherapeutic agents known in theart.

The compositions may be administered orally in effective dosages,depending upon the weight, body surface area, and condition of thesubject being treated. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

In one embodiment, the pharmaceutical compositions may be administeredalone or in combination with other known compositions for treating blooddisorders in a subject, e.g., a mammal. Preferred mammals include cats,dogs, pigs, rats, mice, monkeys, chimpanzees, baboons and humans. In oneembodiment, the subject is suffering from a blood disorder. In anotherembodiment, the subject is at risk of suffering from a blood disorder.

In another embodiment, the pharmaceutical compositions may beadministered alone or in combination with other known compositions fortreating hematological malignancies in a subject, e.g., a mammal.Preferred mammals include cats, dogs, pigs, rats, mice, monkeys,chimpanzees, baboons and humans. In one embodiment, the subject issuffering from a hematological malignancy. In another embodiment, thesubject is at risk of suffering from a hematological malignancy.

The language “in combination with” a known composition is intended toinclude simultaneous administration of the composition of the presentdisclosure and the known composition, administration of the compositionof the present disclosure first, followed by the known composition, andadministration of the known composition first, followed by thecomposition of the present disclosure. Any of the compositions known inthe art for treating blood disorders or hematological malignancies canbe used in the methods of the invention.

The administration of the compositions of the disclosure maybe carriedout in single or multiple doses. For example, the novel compositions ofthis disclosure can be administered advantageously in a wide variety ofdifferent dosage forms, i.e., they may be combined with variouspharmaceutically acceptable inert carriers in the form of tablets,dragees, capsules, lozenges, troches, hard candies, aqueous suspensions,elixirs, syrups, and the like. Such carriers include solid diluents orfillers, sterile aqueous media and various nontoxic organic solvents,etc. Moreover, oral pharmaceutical compositions can be suitablysweetened and/or flavored. In general, the therapeutically-effectivecompounds of this disclosure are present in such dosage forms atconcentration levels ranging from about 5.0% to about 70% byweight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

Sustained release compositions can be formulated including those whereinthe active component is derivatized with differentially degradablecoatings, e.g., by microencapsulation, multiple coatings, etc. In oneembodiment of such a composition of the disclosure, the THU isbio-available about 15 to about 180 minutes before the decitabine. Inanother embodiment, the THU is bio-available about 30 to about 60minutes before the decitabine.

It will be appreciated that the actual preferred amountsof activecompounds used in a given therapy will vary according to the particularcompositions formulated. Optimal administration rates for a givenprotocol of administration can be readily ascertained by those skilledin the art using conventional dosage determination tests conducted withregard to the foregoing guidelines.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of the compounds of thedisclosure generally to ensure their efficacy under normal usecircumstances. Especially when employed for therapeutic treatment ofhumans and animals in vivo, the practitioner should take all sensibleprecautions to avoid conventionally known contradictions and toxiceffects.

The composition, shape, and type of dosage forms of the disclosure willtypically vary depending on their use. This aspect of the disclosurewill be readily apparent to those skilled in the art. See, e.g.,Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing,Eastern Pa.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichthe compound of the disclosure will decompose. Such compounds, which arereferred to herein as “stabilizer” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

The interrelationship of dosages for animals and humans (based onmilligrams per meter squared of body surface) is described in Freireich,et al. 1966 Cancer Chemother Rep 50: 219. Body surface area may beapproximately determined from height and weight of the patient. See,e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N. Y., 1970,537.

Methods of Treatment

In one embodiment, a composition of the disclosure is administered to apatient in need of treatment of a blood disorder. In another embodiment,a composition of the disclosure is administered to a patient in need oftreatment of a hematological or solid malignancy. Other conditions,diseases and disorders that would benefit from such uses are known tothose of skill in the art.

Responsiveness of the disease to compositions of the disclosure can bemeasured directly by comparison against conventional drugs (for example,for hematological or solid malignancies, chemotherapeutics; for certainblood disorders, hydroxyurea, histone deacetylase inhibitors, orerythropoietin), or can be inferred based on an understanding of diseaseetiology and progression. For example, there are a number of HbFexpression assay systems that are widely accepted in the art aspredictive of in vivo effects. Thus, the showing that a compound of thisdisclosure induces HbF expression in these assays is evidence of theclinical utility of these for treating a hemoglobinopathy and/or athalassemia, i.e., a blood disorder.

In one embodiment, “treatment” or “treating” refers to an ameliorationof a hemoglobinopathy and/or a thalassemia, i.e., a blood disorder, orat least one discernible symptom thereof. In another embodiment,“treatment” or “treating” refers to an amelioration of at least onemeasurable physical parameter, not necessarily discernible by thepatient. In yet another embodiment, “treatment” or “treating” refers toinhibiting the progression of a hemoglobinopathy and/or a thalassemia,i.e., a blood disorder, either physically, e.g., stabilization of adiscernible symptom, physiologically, e.g., stabilization of a physicalparameter, or both. In yet another embodiment, “treatment” or “treating”refers to delaying the onset of a hemoglobinopathy and/or a thalassemia,i.e., a blood disorder, or symptoms thereof.

In another embodiment, “treatment” or “treating” refers to anamelioration of a hematological or solid malignancy or at least onediscernible symptom thereof. In another embodiment, “treatment” or“treating” refers to an amelioration of at least one measurable physicalparameter, not necessarily discernible by the patient. In yet anotherembodiment, “treatment” or “treating” refers to inhibiting theprogression of cancer, either physically, e.g., stabilization of adiscernible symptom, physiologically, e.g., stabilization of a physicalparameter, or both. In yet another embodiment, “treatment” or “treating”refers to delaying the onset of a hematological or solid malignancy orsymptoms thereof.

The compositions of the disclosure can be assayed in vitro or in vivo,for the desired therapeutic or prophylactic activity, prior to use inhumans. For example, animal model systems can be used to demonstrate thesafety and efficacy of compounds of this disclosure.

Without wishing to be bound by theory, it is believed thatchedcompositions of this disclosure induce gene expression, for example,fetal hemoglobin expression and, as a result, may be used to treat orprevent a hemoglobinopathy and/or a thalassemia, i.e., a blood disorder.Further without wishing to be bound by theory, it is believed that thecompositions of this disclosure bind to and deplete DNA methyltransferase (specifically, DNMTI), decreasing repression or aberrantrepression of genes that could have therapeutic effects if they wereexpressed, and, as a result, may be used to treat or preventhematological or solid malignancies. It should be noted, however, thatthe compositions might act by a secondary or a different activity, suchas, without limitation, stimulating hematopoiesis, erythropoiesis, andincreasing self-renewal of normal stem cells.

The altered expression of genes could also increase recognition ofmalignant cells by cells of the immune system, whether that immunesystem is the patient's own or an allogeneic immune system reconstitutedthrough allogeneic stem cell transplantation or infusion of donorlymphocytes.

Combination Therapy

The herein-described methods for treating a hemoglobinopathyand/or athalassemia, i.e., a blood disorder, in a subject can further compriseadministering to the subject being administered a composition of thisdisclosure, an effective amount of one or more other therapeutic agents.In one embodiment where another therapeutic agent is administered to asubject, the effective amount of the composition of the disclosure isless than its effective amount would be where the other therapeuticagent is not administered. In another embodiment, the effective amountof the other therapeutic agent is less than its effective amount wouldbe where the composition of the disclosure is not administered.

The herein-described methods for treating a hematological or solidmalignancy in a subject can further comprise administering to thesubject being administered a composition of this disclosure, aneffective amount of one or more other therapeutic agents. In oneembodiment where another therapeutic agent is administered to a subject,the effective amount of the composition of the disclosure is less thanits effective amount would be where the other therapeutic agent is notadministered. In another embodiment, the effective amount of the othertherapeutic agent is less than its effective amount would be where thecomposition of the disclosure is not administered.

In some aspects described herein, the method includes an additionaltherapeutic modality. For example, the additional therapeutic modalityis radiation therapy or a cytotoxic chemotherapy agent, such as ananti-metabolite (e.g., 5-FU, with leucovorin), irinotecan, (or othertopoisomerase inhibitor), doxorubicin, HDAC inhibitors, anti-viralagents, anti-retroviral agents, or any combination all of these agents,including administration of all of these agents. Included withanti-viral agent treatment may be pre-treatment with an agent thatinduces the expression of viral thymidine kinase.

In additional aspects described herein, the methods can includemonitoring the subject for the pharmacodynamic effect of therapy, e.g.,for depletion of DNMTI in normal and malignant cells.

The methods can further include the step of monitoring the subject,e.g., for a reduction in one or more of: a reduction in tumor size;reduction in cancer markers, e.g., levels of cancer specific antigen;reduction in the appearance of new lesions, e.g., in a bone scan; areduction in the appearance of new disease-related symptoms; ordecreased or stabilization of size of soft tissue mass; or any parameterrelated to improvement in clinical outcome. The subject can be monitoredin one or more of the following periods: prior to beginning oftreatment; during the treatment; or after one or more elements of thetreatment have been administered. Monitoring can be used to evaluate theneed for further treatment with the composition of the disclosure or foradditional treatment with additional agents. Generally, a decrease in orstabilization of one or more of the parameters described above isindicative of the improved condition of the subject. Information aboutthe monitoring can be recorded, e.g., in electronic or digital form.

The treatment methods disclosed herein can be used in combination withone or more additional treatment modalities, including, but not limitedto: surgery; radiation therapy, and chemotherapy.

With reference to the methods disclosed herein, the term “combination”refers to the use of one or more additional agents or therapies to treatthe same patient, wherein the use or action of the agents or therapiesoverlap in time. The additional agents or therapies can be administeredat the same time as the composition of the disclosure is administered,or sequentially in any order. Sequential administrations areadministrations that are given at different times. The time betweenadministration of the one agent and another agent can be minutes, hours,days, or weeks.

The additional agent or therapy can also be another anti-cancer agent ortherapy. Non- limiting examples of anti-cancer agents include, e.g.,anti-microtubule agents, topoisomerase inhibitors, antimetabolites,mitotic inhibitors, alkylating agents, intercalating agents, agentscapable of interfering with a signal transduction pathway, agents thatpromote apoptosis, radiation, and antibodies against othertumor-associated antigens (including naked antibodies, immunotoxins andradioconjugates). Examples of the particular classes of anticanceragents are provided in detail as follows: antitubulin/antimicrotubule,e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin,taxotere; topoisomerase I inhibitors, e.g., irinotecan, topotecan,camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin,idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantronehydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU),methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin, alanosine,pyrazofurin, N-Phosphoracetyl-L-Asparate=PALA, pentostatin,5-azacitidine, 5-Aza 2′-deoxycytidine, ara-A, cladribine,5-fluorouridine, FUDR, tiazofurin,N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamicacid; alkylating agents, e.g., cisplatin, carboplatin, mitomycin C, BCNU(Carmustine), melphalan, thiotepa, busulfan, chlorambucil, plicamycin,dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen mustard,uracil mustard, pipobroman, 4-ipomeanol; agents acting via othermechanisms of action, e.g., dihydrolenperone, spiromustine, anddesipeptide; biological response modifiers, e.g., to enhance anti-tumorresponses, such as interferon; apoptotic agents, such as actinomycin D;and anti-hormones, for example anti-estrogens such as tamoxifen or, forexample antiandrogens such as4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide.

Histone deacetylase inhibitors (HDAC inhibitors), a class of compoundsthat interfere with the function of histone deacetylase, are likewisecontemplated as an additional agent for combination therapy. HDACinhibitors include, without limitation, hyroxamic acids (for example,Trichostatin A), cyclic tetrapeptides (for example, trapoxin B),depsipeptides (for example, romidepsin), benzamides, electrophilicketones, aliphatic acid compounds (for example, phenylbutyrate, valproicacid), SAHA/Vorinostat, FK228, Belinostat/PXDIOI, Panobinostat, MS-275,LAQ824/LBH589, C1994, MGCD0103, nicotinamide, NAD derivatives,dihydrocoumarin, naphthopyranone, 2-hydroxynaphthaldehydes,dicarboxamide derivatives, pyridyl and pyrimidinyl derivatives,4-carboxybenzylamino derivatives, fluorinated arylamide derivatives,stilbene-like compounds, 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinonederivatives and phenoxazinone.

A combination therapy can include administering an agent that reducesthe side effects of other therapies. The agent can be an agent thatreduces the side effects of anticancer treatments. A combinationaltherapy can also include administering an agent that reduces thefrequency of administration of other therapies. The agent can be anagent that decreases growth of tumor after the anti-cancer effects ofother therapies have decreased.

Useful combination therapies will be understood and appreciated by thoseof skill in the art. Potential advantages of such combination therapiesinclude the ability to use less of each of the individual activeingredients to minimize toxic side effects, synergistic improvements inefficacy, improved ease of administration or use, and/or reduced overallexpense of compound preparation or formulation. For example, thecompounds of the disclosure may be administered to the subject fortreatment of a hemoglobinopathy and/or a thalassemia, i.e., a blooddisorder, in combination with one or more cytokines. In one embodiment,the cytokine is selected from the group consisting of IL-3, GM-CSF,G-CSF, stem cell factor (SCF) and IL-6.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features. From the above description and the examples thatfollow, one skilled in the art can easily ascertain the essentialcharacteristics of the present invention, and without departing from thespirit and scope thereof, can make various changes and modifications ofthe invention to adapt it to various usages and conditions. For example,the compounds of the disclosure may be used as research tools (forexample, to isolate new targets for performing drug discovery). Thecompounds may, for instance, be radiolabeled for imaging tissue ororgans or be used to form bioconjugates for affinity assays. These andother uses and embodiments of the compounds and compositions of thisdisclosure will be apparent to those of ordinary skill in the art.

The disclosure also encompasses all possible permutations of the claimset, as if they were multiple dependent claims.

Various embodiments of the disclosure could also include permutations ofthe various elements recited in the claims as if each dependent claimwas multiple dependent claim incorporating the limitations of each ofthe preceding dependent claims as well as the independent claims. Suchpermutations are expressly within the scope of this disclosure.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims. All references cited herein are incorporated in theirentirety by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

The description of the present disclosure has been presented forpurposes of illustration and description, but is not intended to beexhaustive or limiting of the invention to the form disclosed. The scopeof the present invention is limited only by the scope of the followingclaims. Many modifications and variations will be apparent to those ofordinary skill in the art. The embodiment described and shown in thefigures was chosen and described in order to best explain the principlesof the invention, the practical application, and to enable others ofordinary skill in the art to understand the invention for variousembodiments with various modifications as are suited to the particularuse contemplated.

The invention is further defined by reference to the following examplesdescribing in detail the preparation of compounds of, the invention. Itwill be apparent to those skilled in the art that many modifications,both to materials and methods, may be practiced without departing fromthe purpose and interest of this invention. The following examples areset forth to assist in understanding the invention and should not beconstrued as specifically limiting the invention described and claimedherein. Such variations of the invention, including the substitution ofall equivalents now known or later developed, which would be within thepurview of those skilled in the art, and changes in formulation or minorchanges in experimental design, are to be considered to fall within thescope of the invention incorporated herein.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1. Decitabine can Deplete DNMTI in Normal Hematopoietic Stem andProgenitor Cells without Causing Measurable DNA Damage or Apoptosis

Decitabine is shown herein to deplete DNMTI without causing measurableDNA damage. Decitabine at a concentration of 0.2-0.5 μM depletes DNMTIin normal hematopoietic precursor cells (data not shown). Normal CD34+hematopoietic precursor cells were isolated from cord-blood. A Q-dotbased immunofluorescence assay quantifies DNMTI depletion by variousdoses of decitabine. Q-dot conjugated 2° ab against anti-DNMTI 1° aballows quantification of DNMTI depletion in normal humanCD34+hematopoietic cells exposed to various levels of decitabine. DNMTIwas quantified in 500 cells for each treatment condition. DAPI was usedto stain the nuclei Image-quant software is used to quantify the DNMTIby the Mean Intensity Fluorescence (MIF) variable. Decitabine levelswere assessed in μM.

FIGS. 1A and B) Decitabine at a concentration of 0.5 μM, added to normalhematopoietic stem and progenitor cells 3×/wk, does not cause measurableDNA damage. However, decitabine 1.0 μM causes measurable DNA damage. A)The normal CD34+ hematopoietic cells subject to the decitabineconcentrations above were assessed for DNA damage by the FastMicro-method for measuring DNA scission 24 hrs after DAC exposure(Riccardi, R., et al. 1982 Cancer Res 42:1736-1739). In contrast,equimolar doses of Ara-C (Ara-C 0.5 μM) and clinically relevant levelsof hydroxyurea (HU 500 μM), cause significant DNA damage (decitabinedose 0.5μM DI, 3, 5; Ara-C dose 0.5 μM D 1, 3, 5; hydroxyurea dose 500μM DI-5). Measurement was performed on D6. B) DNA damage was alsoassessed by phosphorylation (γ) of histone H2AX, an early marker of DNArepair again demonstrating the above concentrations of Ara-C (Ara-C) andclinically relevant levels of HU cause significant DNA damage, but notequimolar amounts of decitabine.

Dark histogram=isotype control for flow-cytometric analysis. Lighthistograms show the γH2AX staining for untreated control cells.Measurement was performed on D6.

FIG. 1C) DAC, at these non-DNA-damaging but DNMTI -depleting levels,produces transient cell-cycle arrest followed by reboundhyperproliferation. Cell cycle status was measured at the varioustimepoints by flow-cytometric assessment of propidium iodide staining.Decitabine treatment causes cytostasis with a rebound increase inS-phase/G2M that occurs approximately 48 h after drug exposure. Resultsare expressed as a percentage of untreated control.

The kinetics of DNMTI depletion and recovery in normal hematopoieticprecursors exposed to a IX addition of DAC 0.5 μM. DNMTI was quantifiedwith a Q dot-based assay. Nuclei were stained with DAPI. Decitabine wasfound to cause minimal or no evidence of apoptosis by flow-cytometricmeasurement of annexin V-FITC and 7AAD double staining (data not shown).Equimolar doses of Ara-C (Ara-C) and clinically relevant levels of HU(Kreis, W., et al. 1991 Leukemia 5:991-998) cause significant apoptosisand cell-death. Measurement was performed on D6.

Therefore, the cytotoxic effects of decitabine can be separated from itsDNMTI depleting effects at concentrations between 0.2-0.5 μM. Therefore,for non-cytotoxic epigenetic therapy, the pharmacologic goal is toincrease time above threshold concentration required to deplete DNMT(approximately 0.2 μM) while avoiding high peak levels that cause DNAdamage (>0.5-1.0 μM).

Example 2. DNMTI -Depleting but Non-DNA Damaging, Doses of Decitabinehave Opposite Effects on Normal Stem-Cells (HSC) Versus Leukemia Cells

CD34 cells transduced with AMLI-ETO recapitulated some features ofleukemia stem-cells (LSC) (impaired differentiation and increasedself-renewal) and represented a first-hit or early model of leukemictransformation. The Kasumi-1 cell-line is derived from a patient withAMLI-ETO leukemia and represents a late-stage model of transformationand malignant evolution. Both of these models terminally differentiatewith non-DNA-damaging but DNMTI -depleting doses of decitabine. FIG. 2A)Cell counts: an ideal therapeutic index was seen, with proliferation ofnormal cells while leukemia cells decline. In contrast, standard therapy(Ara-C), given at equimolar doses was more devastating to normal cells.Normal cells retained primitive morphology with decitabine treatment(data not shown). In contrast, CD34 AMLI-ETO and Kasumi-1 cellsmorphologically differentiated (decreased nuclear cytoplasmic ratio,nuclear segmentation or condensation, cytoplasmic granulation andvacuolation). FIG. 2B) Normal CD34+ cells treated with decitabinedemonstrated increased self-renewal (Milhem, M., et al. 2004 Blood103:4102-4110), therefore, decitabine treatment maintains colony-formingability. In contrast, CD34 AMLI-ETO cells (leukemia cells) terminallydifferentiate with decitabine treatment and colony forming ability isabrogated (data not shown).

Example 3. DNMTI Depletion without DNA Damage Produces TerminalDifferentiation (not Immediate Apoptosis) of Primary Leukemia Cells fromPatients

Primary leukemia cells obtained from bone marrow or peripheral blood(with informed consent on an IRB approved protocol) were cultured inmedia supplemented with cytokines with or without decitabine 0.5 μMadded 2×/week. These concentrations of decitabine did not cause DNAdamage or immediate apoptosis (FIG. 3A). The samples were obtained froma spectrum of AML sub-types. FIG. 3B) Cell counts at D7 in untreatedcontrol versus decitabine-treated cells. Giemsa staining demonstratedterminal myelomonocytic differentiation of decitabine treated cells in12 of 14 cases (data not shown). In cases of resistance, DNMTIexpression in the cells was retained, indicating failure of decitabineactivity, rather than resistance to the effects of DNMTI depletion (datanot shown).

Decitabine 0.5 μM, concentrations that did not cause earlyapoptosis asmeasured by Annexin staining, terminally differentiated solid tumorcell-lines (renal cancer, small cell lung cancer, hepatocellular cancer,prostate cancer, bladder cancer), producing morphologic changes ofdifferentiation (increased cell-size, decreased nuclear-cytoplasmicratio). Melanoma is a cancer that is resistant to conventionalapoptosis-based therapy. Decitabine 0.5 μM induced changes of terminaldifferentiation in 7 melanoma cell lines. FIG. 4) Cell counts in controland decitabine treated cells at D9. Giemsa-stained control and treatedcells at D8—morphologic changes indicated that decitabine treatmentinduced differentiation (data not shown).

Example 4. A Differentiation Therapy Regimen of Decitabine is Effectiveand Very Well Tolerated in a Xeno-Transplant Model of Avastin-ResistantRenal Cancer

In an effort to apply the proposed formulation and regimen to treatdifferent cancer histologies, a renal cell cancer cell-line (Reno-1) wasdeveloped from a surgical sample of renal cancer and used in axeno-transplantation experiment. In a xenograft model of resistant renalcancer, SQ decitabine given 3×/week at a dose of Img/ni2 (starting onDay 9—tumor vol. 100) significantly decreased tumor volume withoutevidence of toxicity in the mice (no change in weight, appearance orblood counts). This tumor was relatively resistant to avastin andsunitinib, standard agents used to treat renal cancer. FIG. 5A)Decitabine (DAC) decreased tumor volume (p<0.001, t-test), compared tocontrol (PBS) or Sunitinib. Sunitinib antagonized the effect ofdecitabine, presumably by cytostasis that decreased the S-phasedependent incorporation of decitabine into tumor. H&E staining ofparaffin embedded sections demonstrated necrosis in thedecitabine-treated samples (data not shown). The percentage of necrosisin each tissue section was evaluated in a blinded fashion. ThePBS-treated animals (controls) had dense, healthy tissue which is poorlystaining (grey tissue highlighted with white arrows). FIG. 5B)Paraffin-embedded sections do not lend themselves to analysis forcytological detail. Therefore, the morphology of Giemsa-stained Reno-1cells cultured with decitabine was examined in vitro. Decitabine-treatedcells increase in size and demonstrate prominent nuclear chromatinclumps.

Example 5. The PUER Model of Pu.1-Mediated Hematopoietic DifferentiationProvides an Insight into the Mechanisms that Underlie the OppositeEffects of DNMTI Depletion on the Self-Renewal of Normal HSC VersusLeukemic Cells

Terminal differentiation is critically dependenton lineage-determiningtranscription factors such as PU.1. PU.1, like other lineage-determiningDNA binding factors, demonstrates both transcription repression andtranscription-activating functions, determined by interactions witheither corepressors versus coactivators. The murine PUER cell-line isderived from Pu.1 knock-out cells, which have been transduced with aretroviral vector which expresses Pu.1 fused to the estrogen-receptor.Addition of the estrogen agonist tamoxifen (OHT) to these cells causesPu.1 to be functionally reintroduced into the cell through translocationinto the nucleus, and triggers terminal differentiation.

FIG. 6) Pu.1 induced terminal differentiation involves orderly andsequential repression of genes associated with self-renewal (HoxB4,Bmi-1, c-Kit), followed by activation of genes associated with terminaldifferentiation (Mcsfr, Gmcsfr, F4/80) (latter data not shown).

Example 6. The Phenotypic Consequences of DNMTI Depletion Depend on theDifferentiation Chronology of the Cell

FIG. 7A) The effect of decitabine on proliferation of PUER is dependenton the timing of decitabine addition in relationship to Pu.1 activation(Pu.1 is functionally activated by adding OHT to the cells). Addingdecitabine concurrent with Pu.1 activation impaired Pu.1-mediatedterminal differentiation and preserved some cell proliferation. However,adding decitabine 6h after Pu.1 activation did not. FIG. 7B) Concurrentdecitabine and Pu.1 activation inhibited differentiation, but decitabine6 hrs after Pu.1 activation did not. Flow cytometry was used to measureF4/80 as a marker of terminal macrophage differentiation and c- Kit asmarker of stem-cells. Cell morphology was consistent with theflow-cytometry data (data not shown). FIG. 7C) Decitabine additionconcurrent with Pu.1 activation prevented the first step in terminaldifferentiation of pro-self-renewal gene repression. However, decitabineaddition after pro-self-renewal gene repression had occurred (6 hoursafter OHT) increased pro-differentiation gene expression.

Therefore, the phenotypic consequences of DNMTI depletion criticallydepend on the differentiation chronology of the cell. By preventing thefirst step in differentiation, which is repression of pro- self-renewalgenes, decitabine can increase self-renewal even in adifferentiation-promoting context. These findings are a reasonableexplanation for our published observation that decitabine increasesself-renewal of normal HSC (Milhem, et al. 2004 Blood 103(11):4102-10).In contrast, increased differentiation in response to decitabine that isadded after self-renewal genes have already been repressed, resemblesthe effect of decitabine on leukemia cells. The opposite effects ofdecitabine on HSC versus leukemia cells could be proposed to resultbecause leukemia is arrested differentiation in progress, distinct fromself-renewal of HSC.

Example 7. Molecular Events when a Leukemia First-Hit Event (Disruptionof Runxl) Inhibits Pu.1 Mediated-Differentiation

Runxl disruption, by congenital or acquired Runxl mutations andchromosome translocations, is one of the most frequent genetic events inmyelodysplasia and leukemia.

Knock-down of Runxl expression in PUER cells did not preventPu.1-mediated repression of pro- self-renewal genes, but did inhibitPu.1-mediated activation of pro-differentiation genes. Using lenti-viralshRNA delivery, Runxl expression was knocked down in PUER cells, anddecreased Runxl expression was confirmed by Western blot (between 25-50%of control levels), in different PUERshRunxl clones (data not shown).PUERshRunxl cells did not terminally differentiate in response to Pu.1activation (+OHT) (data not shown). FIG. 8) In PUERshRunxl cells, thefirst-step in Pu.1 mediated differentiation of pro- self renewal (Bmi-1,HoxB4, c-Kit) repression is intact. However, Runxl knock-down preventedthe next chronological step in differentiation—activation ofpro-differentiation genes (F4/80, mcsfr, gmcsfr).

Example 8. Leukemia Cells from Patients Conform to the Model

To examine the clinical relevance of the above findings regarding arrestin differentiation-transit, the levels of lineage-specifying factorswere measured in primary leukemia cells from patients. Keylineage-specifying transcription factors in hematopoiesis include PU.1(required for macrophage and B-lymphocyte production), CEBPα and CEB Pϵrequired for neutrophil production and GATAI for erythroid production(Iwasaki, H., et al. 2006 Genes Dev 20:3010-3021). The levels of thesefactors increase during differentiation into the respective lineages.The expression levels of these factors were measured in bone marrowaspirate cells from normal donors, patients with low-riskmyelodysplastic syndrome (MDS—a clonal hematologic disorder which oftenprecedes AML) and patients with high-risk myelodysplastic syndrome andacute myeloid leukemia (high-risk disease). The low-risk MDS patientbone marrow aspirates contain abnormally differentiated and increasedimmature cells, but <5% myeloblasts. In the high-risk patient bonemarrow aspirates, the average percentage of myeloblasts was 40%. Thehigh-risk samples, although morphologically the least mature, had thehighest levels of the myeloid lineage-specifying factors CEBPα, CEB Pϵand PU.1 (FIGS. 9A-9B). GATAI levels were not significantly increased inthe disease samples compared to normal controls. The clinical annotationof the samples analyzed is shown (FIG. 9C).

Micro-array gene-expression data from 54 AML patients demonstrated asimilar pattern of increased expression of lineage-specifying factors inAML samples compared to normal bone marrow cells (Yagi, T., et al. 2003Blood 102:1849-1856).

Unlike DNA mutation or chromosome aberration, it is not immediatelyobvious that a difference in promoter CpG methylation between amalignant and a normal sample is abnormal, since promoter CpGmethylation varies among different tissues and with stage ofdifferentiation. Therefore, classifying promoter CpG sites by themethylation changes that occur during normal differentiation couldimprove interpretation of DNA methylation studies in cancer andleukemia. In particular, such classification could illuminate the roleof differentiation in malignant cell-specific patterns of promoter CpGmethylation.

To address this issue, a promoter CpG methylation micro-array was usedto classify CpG sites by hypo-methylation, hyper-methylation, or nosignificant methylation change during normal myeloid differentiation.This classification was applied to a study of promoter CpG methylationpatterns in primary myelodysplastic syndrome (MDS) and acute myeloidleukemia (AML) cells. In primary MDS cells, methylation atdifferentiation responsive CpGs was the inverse of that in normal CD34+hematopoietic stem and progenitor cells (HSPC), with hypo-methylation ofCpG that are normally hyper-methylated in CD34+ HSPC and vice-versa. Inhigh risk (>5% myeloblasts) MDS/AML samples, and AML cell linesincluding CD34+ AML cell lines, this pattern was further exaggerated.This difference between normal HSPC and MDS/AML cell epigenetics couldcontribute to contrasting differentiation fates in response to drugsthat inhibit chromatin-modifying enzymes (FIG. 9D).

This observation provides insight into leukemogenesis and MDS/AMLbiology. Promoter CpG methylation reflects differentiation stage orcontext. The pattern of promoter CpG methylation in the MDS/AML cells isconsistent with a differentiation context that is more advanced orcommitted than normal HSPC. This possibility is clarified by the highexpression of the key lineage- specifying transcription factors PU.1 andCEBPα in MDS/AML cells compared to normal hematopoietic stem andprogenitor cells. Therefore, the pattern of promoter CpG methylationsuggests, and is likely one dimension of, a lineage-committeddifferentiation context of MDS/AML cells.

FIGS. 9A-9D. Leukemia cells from patients are differentiation-impairedafter lineage-commitment. FIGS. 9A-9B) High risk MDS and AML cellsexpress high levels of lineage- specifying factors (measured by RQ-PCR).Samples are bone marrow from normals, patients with low risk MDS (<5%myeloblasts), and high risk MDS/AML (>5% myeloblasts). FIG. 9C) WHOclassification and detected chromosome abnormalities in the analyzedsamples. FIG. 9D) In MDS and AML bone marrow, the direction ofmethylation change at differentiation responsive CpG sites mimics thatseen during normal differentiation, only exaggerated.

Methylation levels are represented by a β-value between 0 (unmethylated)and 1 (fully methylated). Promoter CpG sites were classified into 3categories: CpG sites that undergo a significant (p<0.001, t-test)increase in methylation from normal stem and progenitor cells (HSPC,NCD34+) to normal mature cells (NBM) (108 CpG, ‘hypomet. in NCD34+’)(left box plots); CpG sites that undergo a significant (p<0.001, t-test)decrease in methylation from NCD34+ to NBM (162 CpG, ‘hypermet. inNCD34+’) (middle box plots); CpG sites that do not undergo a change inmethylation between NCD34+ and NBM (1236 CpG, ‘no met. change during n.diffn.’) (right box plots). Asterixes represent statisticallysignificant differences between the median in the sample group comparedto the NBM group (Kruskal-Wallis). Actual p-values are: ‘Hypo-met inNCD34’—NBM v NCD34 <0.0001, NBM v LoRisk <0.0001, NBM v HiRisk <0.0001.‘Hyper-met in NCD34’—NBM v NCD34 <0.0001, NBM v LoRisk=0.024, NBM vHiRisk <0.0001. ‘No Change’—NBM v NCD34 <0.0001#, NBM v LoRisk NBM vLoRisk=0.024, NBM v HiRisk <0.0001, NBM v HiRisk <0.0001. NCD34=CD34+cells isolated from normal bone marrow (n=9), NBM=normal whole bonemarrow (n=42), LoRisk=bone marrow from low-risk MDS patients (n=27),HiRisk=bone marrow from high-risk MDS/AML patients (n=130). Box -plotboundaries=inter-quartile range, horizontal line=median, ‘+’=mean,whiskers=range of values, small boxes=out- lying values. #CpG wereclassified as ‘no change in met’ based on a t-test to compare meansbetween NCD34 and NBM, whereas a Kruskal-Wallis test to compare mediansis used here.

Example 9. A Model of Normal Differentiation and Leukemia Self-Renewalthat Explains why DNMTI Depletion has Opposite Effects on Normal andMalignant Cells

Differentiation mediated by a lineage-specifying transcription factor orby cytokines (Milhem, M., et al. 2004 Blood 103:4102-4110) requiresorderly repression of stem-cell associated genes followed byupregulation of differentiation-fate associated genes. The repression ofstem-cell associated genes requires chromatin modifying proteins such asDNMTI. Therefore, DNMTI depletion, by preventing this initial phase,prevents differentiation and maintains self-renewal of dividing normalstem-cells, even in differentiation inducing conditions (Milhem, M., etal. 2004 Blood 103:4102-4110) (data not shown).

In a substantial number of AML cases, differentiation arrest occursafter lineage-commitment (hence AML cells express lineage markers andhigh levels of lineage-specifying factors) and after the phase ofstem-cell associated gene repression. Therefore, the self-renewal(proliferation at the same level of differentiation) of the leukemiacells is an aberrant persistence of the cell-division of differentiatingcells, which is usually terminated by completion of the differentiationprocess. The differentiation impairment which maintains this abnormalself-renewal, although it may be initiated by genetic abnormalities, isfinally mediated by epigenetic mechanisms that aberrantly repress genesnecessary for differentiation. Therefore, DNMTI depletion to antagonizethe transcription repression machinery restores the differentiation forwhich these cells are poised and thereby terminates the abnormalself-renewal. Observations in cancer cell lines representing a spectrumof cancer histologies indicate that the proposed model is relevant inmany cancers.

Example 10. Translation of these In Vitro Observations into Effective InVivo Therapy Must Overcome a Number of Pharmacologic Obstacles

The MA9 xenograft model of human leukemia was treated withintraperitoneal decitabine alone (1 mg/m²3×/week). Decitabine increasedsurvival by approximately 20%, but all mice succumbed to leukemia (FIG.10). This poor result, which did not reflect the in vitro findings,highlights the pharmacologic barriers that limit the in vivo activity ofdecitabine and hinder successful translation of very promising in vitrofindings. CDA is the most important pharmacologic barrier to successfultranslation of in vitro findings into in vivo therapy. The inventionaddresses this barrier to effective clinical translation of the in vitroobservations.

An antagonist of the decitabine degrading enzyme cytidine deaminase(CDA)- tetrahydrouridine (THU). THU is a pyrimidine analogue thatinhibits CDA (Ki 3−5×10−8M). THU has a benign toxicity profile,well-characterized PK, and has been administered to humans byintravenous (IV), sub-cutaneous (SQ), and oral (PO) routes in a numberof clinical trials (Ho, D. H., et al. 1978 J Clin Pharmacol 18:259-265;Kreis, W., et al. 1977 Cancer Treat Rep 61:1347-1353; Kirch, H. C., etal. 1998 Exp Hematol 26:421-425; Yue, L., et al. 2003 Pharmacogenetics13:29-38; Gilbert, J. A., et al. 2006 Clin Cancer Res 12:1794-1803;Bhojwani, D., et al. 2006 Blood 108:711-717; Liu, Z., et al. 2007Nucleic Acids Res 35:e31).

Example 11. Pharmacologic Obstacle: Inter-Individual Variation inResponse to Cytosine Analogues

The AA genotype of CDA is associated with poor outcome in response tonucleoside analogue therapy. In humans, CDA is subject to anon-synonymous single nucleotide polymorphisms (SNP) (RS2072671) whichproduces an A→C transition in the ancestral allele, changes lysine toglutamine at amino-acid position 27 and decreases CDA activity 3-fold(Gilbert, J. A., et al. 2006 Clin Cancer Res 12, 1794-1803; Kirch, H. C.et al. 1998 Exp Hematol 26, 421-425; Yue, L., et al. 2003Pharmacogenetics 13, 29-38). In the human hapmap (publicly availablehaplotype map of the human genome), 40% of Caucasians are homozygouswith the AA genotype, 50% heterozygous with AC genotype and 10%homozygous with CC genotype (AA frequency is >90% in Asians andAfricans). Since CDA can mediate cancer resistance to cytosineanalogues, it was examined whether these different genotypes of CDApredicted clinical outcomes in MDS and AML patients treated withcytosine arabinoside (ARA-C), 5-azacytidine, or decitabine.

The AA genotype of CDA, which has greater enzymaticactivity, isassociated with early relapse during cytosine analogue therapy but notwith non-cytosine analogue therapy. It was hypothesized that the moreenzymatically active AA genotype of CDA, by further limiting theactivity of cytosine analogues (ARA-C, 5-azacytidine or decitabine),could cause primary resistance or early relapse (relapse within 2 years)in MDS or AML patients treated with these drugs, but not in MDS or AMLpatients not being treated with cytosine analogues. Using the HumanNS-12 BeadChip (Illumina, San Diego, Calif.) and allele-specific DNAsequencing, 24 month survival was stratified by CDA genotype in 81 MDSand AML patients treated at the Cleveland Clinic. As expected, the AAgenotype was associated with decreased 24 month survival (p=0.05 Cox) inpatients treated with cytosine analogues (data not shown). Numbers forthe CC genotype are small, but it could be proposed that CC genotype mayincrease mortality from toxicity, but decrease relapse mortality. Inpatients who were not treated with cytosine analogues (these patientsreceived arsenic or gemtuzumab), CDA genotype did not influence survivaloutcome (data not shown).

This data may explain the poorer leukemia outcomes seen in non-Caucasianpopulations (who are>90% AA genotype), and indicates that thecombination decitabine -tetrahydrouridine (THU) agent contemplatedherein should be a significant advance in MDS and AML therapy (andpossibly therapy for other cancers) in all populations, since byinhibiting CDA, it is likely to address a major mechanism that underliespoor outcome with cytosine analogues.

Early relapse of leukemia is believed to represent selection forchemo-resistant clones, whereas late relapse may represent the emergenceand evolution of leukemia stem-cells that were quiescent and unexposedto induction and consolidation chemotherapy. The combination ofdecitabine with THU has the potential to address both forms of relapse.Early relapse can be reduced by improving the pharmacologic profile(increasing time above threshold concentration, decreasinginter-individual variation) of decitabine. The lack of toxicity of theproposed approach, which should facilitate chronic, long-term therapy,may address late relapse, since even relatively quiescent LSC should atsome point be exposed to chronically administered decitabine.

Example 12. Pharmacologic Obstacle: The Very Brief In Vivo Half-Life ofDecitabine

As seen above, CDA genotype is associated with significantly differentoutcomes in response to cytosine analogue therapy. This reflects thevery large influence CDA has on cytosine analogue therapy in vivo: (i)Because of destruction by the enzyme CDA, the in vivo half-life of IVdecitabine is <20 minutes, compared to an in vitro half-life of 5-9hours (Liu, Z., et al. 2006 Rapid Common Mass Spectrum 20: 1117-1126).In order to exploit decitabine's unique quality amongst nucleosideanalogues, its ability to deplete DNMTI at low concentrations, thepharmacologic objective of therapy is to maximize time-above-thresholdconcentration for depleting DNMTI (>0.2 μM), while avoiding the highpeak levels (>1 μM) that damage DNA (the DNA damage that occurs athigher levels causes toxicity that limits the cumulative dose of drugand is, therefore, counter-productive). To examine how different routesof administration might serve this pharmacologic objective, decitabinepharmacokinetics were studied in the non-human primate baboon model.

FIG. 11: Subcutaneous (SQ) and oral decitabine produces lower peaklevels but a longer time-above-threshold for DNMTI depletion (˜0.2 μM)than IV decitabine. Baboons were treated with IV decitabine 0.5 mg/kg (2animals), SQ decitabine 0.5 mg/kg (2 animals), or oral decitabine 10mg/kg (2 animals). Blood was collected for pharmacokinetic (PK) analysisat 7 time-points per animal and decitabine levels measured by LC-MS(Liu, Z., et al. 2006 Rapid Common Mass Spectrum 20:1117-1126). Thisdata demonstrates: (i) SQ and oral administration can produce a longertime-above threshold concentration (˜0.2 μM) for depleting DNMTI than IVdecitabine. Indeed, intravenous (IV) decitabine has an abbreviatedhalf-life of <20 mins; (ii) IV administration, but not SQ or oral, canproduce peak levels (>1 μM) associated with DNA damage; (iii)Significant inter-individual variability in PK that is most prominentwith oral administration of the drug.

Example 13. Pharmacologic Obstacle: Malignant Cell Resistance toDecitabine is Pharmacological Rather than Biological

Resistance to the effects of decitabine appears to be pharmacologicrather than biological, i.e., resistance is associated with a failure ofdecitabine to deplete DNMTI, rather than continued proliferation despitedepletion of DNMTI.

FIGS. 12A and B) Using the Q-Dot immunofluorescence assay for DNMTIlevels, decitabine-sensitive cell lines demonstrate DNMTI depletion fromthe nucleus (in comparison with decitabine-resistant cell lines).However, the decitabine-resistant cell-line PC3 demonstrated persistentDNMTI expression. This indicates that the mechanism of resistanceinvolves decreased decitabine entry into the cell, increased decitabinedestruction CDA, or decreased activation by DCK.

Example 14. Non-Clinical Pharmacokinetics of Decitabine Alone and inCombination with THU

Pharmacokinetics of decitabine in non-human primates, poor oralbioavailability of decitabine in non-human primates: thepharmacokinetics of decitabine were assessed in two baboons followingoral, SQ and intravenous administration. This experiment demonstratedthat the oral bioavailability of decitabine in these two baboons rangedfrom approximately 0.2% to 7.0% (FIG. 14). Decitabine was administeredto two baboons IV (0.5 mg/kg), SQ (0.5 mg/kg) and orally (10 mg/kg).Blood was collected at 7 time-points and plasma concentrations weredetermined using an LC/MS/MS method adapted from a previously publishedmethod 26. This data demonstrates: (i) that SQ and oral administrationcan produce a longer time-above-threshold concentration (˜0.2 μM) fordepleting DNMTI than IV decitabine; (ii) that at the doses studied, IVadministration, but not SQ or oral, can produce peak levels (>1 μM)associated with DNA damage; and (iii) significant inter-individualvariability in decitabine exposure that is most prominent with oraladministration of the drug.

Example 15. Substantial Inter-Individual Variation in OralBio-Availability of Decitabine in Baboons

Inter-individual variability was likewise seen in seven baboons thatreceived 10 mg/kg decitabine orally, with the PK being measured at 6 or7 time-points (FIG. 15).

Example 16. Administration of Oral THU Prior to Oraldecitabine Addressesthe Substantial Inter-Individual Variability in Oral Bio-Availability ofDecitabine

High and low responder baboons (PA7470 and PA7484, respectively) weregiven 10 mg/kg decitabine alone, 0.5 mg/kg decitabine and 20 mg/kgtetrahydrouridine (THU) concurrently, or 0.5, 2, or 5 mg/kg decitabine60 minutes after 20 mg/kg tetrahydrouridine (THU). Cmax (ng/mL), Tmax(min), and AUClast (min*ng/mL) were measured and are shown in Table 1,below, and FIG. 16. Following the oral administration of drugs, theplasma concentration generally reaches, in principle, a single,well-defined peak (Cmax) at the time of Tmax. AUClast refers to the areaunder a plotted plasma concentration-time curve (not shown) at the lastrecorded timepoint.

TABLE 1 Cmax Tmax AUClast Animal Decitabine (mg(kg) (ng/mL) (min)(min*ng/mL) PA7470 10 mg/kg 51.59 60 6278.775 PA7470 0.5 mg/kg + 20mg/kg 4.24 30 3472.5 THU (0 min) PA7470 0.5 mg/kg + 20 mg/kg 3.06 66356.81 THU (60 min) PA7470 5 mg/kg + 20 mg/kg 48.54 189 7534.665 THU (60min) P47484 10 mg/kg 2.33 45 190.35 PA7484 2 mg/kg + 20 mg/kg 34.01 1801729.15 THU (60 min) P47484 5 mg/kg + 20 mg/kg 65.87 120 5621.05 THU (60min)Of note, the administration of THU and decitabine increases the oralbioavailability of decitabine in the poor responder baboon and convergesthe PK between poor and good responder baboons (4^(th) and 7^(th) valuesin final column) in comparison with decitabine alone (1^(st) and 5^(th)values in final column). In effect, combining THU with oral decitabineincreased the exposure of decitabine approximately 60-fold in an animalwith relatively low bioavailability and approximately 2-fold in ananimal with relatively high bioavailability, such that the extensiveinter-individual variability in decitabine exposure was substantiallyreduced.

Example 17: Identifying the Dose of THU to use to Increase OralBioavailability

Baboons received varying doses (2 mg/kg or 20 mg/kg) oftetrahydrouridine 60 minutes before a dose (5 mg/kg) of decitabine. Twobaboons (PA7484 and PA7470) were administered oral THU 20mg/kg (400mg/m²) 60 minutes prior to oral decitabine 5 mg/kg, or decitabine alone.After a wash-out period of greater than 2 weeks, these same two animalswere administered oral THU 2 mg/kg (50 mg/m²) 60 minutes prior to oraldecitabine 5 mg/kg, or oral decitabine alone. Unlike 20 mg/kg (500mg/m²), 2 mg/kg (50 mg/m²) of THU was insufficient to achieve the targetplasma concentration of decitabine of >50 ng/mL (FIG. 17). Both doses oftetrahydrouridine significantly increased decitabine oralbio-availability, but this increase was significantly greater with thehigher dose (500 mg/m², or, 20 mg/kg) of tetrahydrouridine.

Example 18. Identifying the Optimal Timing Between Oral THU and OralDecitabine Administration (Oral Bio-Availability of Decitabine UponConcurrent vs. Prior Administration of Tetrahydrouridine)

High-responder and low-responder baboons each received either 20 mg/kg(500 mg/m²) tetrahydrouridine (THU) orally 60 minutes before oraladministration of 5 mg/kg decitabine or concurrently with thedecitabine. Decitabine levels were measured at various timepoints byLC-MS.

Notably, when the tetrahydrouridine was given orally concurrently withthe decitabine, the oral bio-availability of the latter exhibited asignificant drop-off in comparison to administration of thetetrahydrouridine 60 minutes prior to decitabine (data not shown).

Example 19: Confirmation that Oral THU Increases the OralBio-Availability of Decitabine Approximately 4-Fold, and Decreases theSubstantial Inter-Individual Variation in Decitabine OralBio-Availability

In seven baboons, the administration of THU prior to decitabineincreased the oral bioavailability of decitabine approximately 4-foldand decreased inter-individual variation in decitabine pharmacokinetics.Seven baboons were treated with decitabine alone 10 mg/kg by oralgavage, or after a wash-out period of at least 2 weeks, with THU 20mg/kg by oral gavage followed by decitabine 5 mg/kg by oral gavage. In 7animals administered oral decitabine alone at 10mg/kg, the mean AUC was1604 and the median AUC was 463 min*ng/ml (FIG. 18A). In these same 7animals administered THU 20 mg/kg followed by half the dose ofdecitabine (5 mg/kg), the mean AUC was 2820.914 and the median AUC was2284 min*ng/ml (FIG. 18A). Therefore, administering oral THU prior tooral decitabine produced a fold- increase in oral bioavailability ofapproximately 10-fold when considering medians, and approximately3.5-fold when considering means. The largest increases in AUC withco-administration were seen in the animals with low AUCs with decitabinealone (FIG. 18B). Therefore, the large inter- individual variation seenwith decitabine alone, represented by the separation between mean andmedian AUC, was substantially dampened by coadministration of THU withdecitabine (FIG. 18A).

Mice received varying doses (30 mg/kg, 15 mg/kg, and 7.5 mg/kg) oftetrahydrouridine 30 minutes before a dose (16 mg/kg) of decitabine.Decitabine levels were measured by LC-MS. 50 mg/m tetrahydrouridineadministered prior to the administration of decitabine resulted in anapproximately 5-fold (certainly pharmacologically significant) increasein decitabine oral bio-availability (data not shown). A slight drop-offwas observed in decitabine oral bio- availability measured when thedosage of THU was decreased from 50 to 25 mg/m.

Of note, a similar cytidine deaminase expression pattern is found inhumans and mice (data not shown). This gene expression data can beobtained from the public gene expression database GenAtlas.

Example 20. Phase ½ Study of Chronic Low Dose IVAdministration ofDecitabine in SCD

Based on previously conducted trials, in which no clinically significantadverse events occurred, a chronic administration study was conducted toidentify the toxicity and effectiveness of repeated decitabine dosingover 36 weeks (9 months) in 7 subjects with HU refractory SCD (DeSimone,J., et al. 2002 Blood 99:3905-3908). Decitabine was administered by I.V. infusion at 0.3 mg/kg/day, 5 consecutive days per week for 2 weeks,followed by a 4-week observation period. If the absolute neutrophilcount (ANC) dropped below 1×109/L, the dose was reduced by 0.05 g/kg/dayin the next 6-week cycle. An optimal drug dose was obtained for eachsubject, and resulted in an elevated HbF without neutropenia (ANC nadir>1.5×109/L).

Pharmacodynamic effects: the average HbF and average maximal HbF levelsattained during the last twenty weeks of treatment for the 7 SCDsubjects were 13.93±2.35% and 18.35%±4.46%, respectively (from abase-line of 3.12%±2.75%).

The average and average maximal hemoglobin values were 8.81±0.42 g/dLand 9.7±0.53 g/dL, respectively (from a base-line of 7.23±2.35 g/dL)(Table 2, below).

TABLE 2 Hemoglobin and HbF levels before and during the last 20 of 36wks of treatment with decitabine HbF (%) Total Hemoglobin (g/dL) SubjectPre Avg Max Pre Avg Max 1 0.8 12.40 ± 1.25 14.4 6.2 9.05 ± 0.48 9.6 26.8 14.55 ± 1.32 16.3 8.2 9.37 ± 0.60 10.3 3 1.4 12.75 ± 2.28 17.2 6.08.34 ± 0.55 9.5 4 0.6 10.30 ± 2.05 14.4 7.2 8.28 ± 0.52 9.0 5 2.9 16.42± 2.31 24.6 8.0 8.91 ± 0.57 9.6 7 6.2 16.70 ± 2.55 23.2 7.8 8.92 ± 0.7910.4 Mean ± 3.12 ± 13.93 ± 2.35 18.35 ± 7.32 ± 8.81 ± 0.42 9.73 ± SD2.75 4.46 0.94 0.53

Individual maximal F-cell numbers during the trial ranged from 58-87%(i.e., an average over all 7 subjects of 69±10.12%).

Hematologic side-effects and toxicity: despite periodic depressions inANCs, which occurred 5 to 6 weeks after beginning each treatment cycle,the average ANC during the last 20 weeks of treatment (4.2±1.35×109/L)was not significantly different from the pretreatment average(4.6±1.56×109/L). The ANCs of two HU non-responder subjects never fellbelow 2.0×109/L and the nadirs, which occurred at 5-6 weeks of eachcycle, generally remained above 3.0×109/L. No clinical sequelae of bloodcount changes occurred.

Non-hematologic toxicity: no non-hematologic toxicity occurred. Patientsdid not require anti-emetics.

Example 21. Phase ½ Study of SQ Administration of Decitabine

A Phase ½ study was initiated using decitabine given by the SQ route inorder to assess the safety of decitabine given by the SQ route, toproduce cumulative increases in fetal and total hemoglobin throughweekly administration, and to explore the mechanism by which decitabineincreases HbF (Saunthararajah, Y., et al. 2003 Blood 102:3865-3870).Eight subjects with multiple clinically significant complications of SCDwere treated. Decitabine was administered at 0.2 mg/kg SQ 1 to 3 timesper week in 2 cycles of 6-week duration with a 2-week interval betweencycles. In cycle 1, drug was administered twice per week on 2consecutive days. If the patient achieved an F-cell percentage (%F-cells) of at least 80% during cycle 1, the dose frequency was reducedto once per week in cycle 2. If the highest % F-cells during the cycle 1was less than 80%, the dose frequency was increased to 3 times per weekin cycle 2.

Pharmacodynamic effects: all subjectsdemonstrated statisticallysignificant increases in HbF expression (FIG. 13). DNA methylationanalysis of the γ-globin promoter in DNA isolated from bone marrowaspirate cells was demonstrated post-treatment hypomethylation at thislocus (data not shown). At higher doses, decitabine is known to becytotoxic. To determine if this dose and schedule of decitabine wascytotoxic, bone marrow morphology was evaluated by independent andblinded hematopathology review of pre-treatment and post-treatment bonemarrow aspirates. There was no decrease in bone marrow cellularity. Anincrease in erythroid cells and megakaryocytes was noted (data notshown). Flow cytometric analysis of propidium iodine stained freshmarrow aspirate cells did not demonstrate an increase in the sub-GIapoptotic fraction (data not shown).

Hematologic side-effects and toxicity: one patient had (National CancerInstitute/Cancer Therapy Evaluation Program (NCI/CTEP) grade 4neutropenia (nadir ANC 0.4×103/μl), two had grade 3 neutropenia (nadirANC 0.8×103/μl). Neutropenia recovered within 7 days of the lastdecitabine dose. Neutropenic fever did not occur. Platelet countsincreased in all subjects during treatment. The highest platelet countwas 877×109/L. No clinical sequelae to these blood count changesoccurred.

There was an inverse relationship between platelet and neutrophil counts(data not shown). The changes in bone marrow morphology and in vitrostudies with non-cytotoxic levels of decitabine indicate that themechanism of decreased neutrophils and concurrent increased platelets isaltered hematopoietic stem cell differentiation (Saunthararajah, Y., etal. 2003 Blood 102:3865-3870).

Four patients consented to serial bone marrow aspirate and biopsyanalysis before and each 6-wk cycle of decitabine treatment.

TABLE 3 Results of bone marrow aspirate and biopsy analysis.Pre-Treatment After Cycle 1 After Cycle 2 M:E MK/ M:E MK/ M:E MK/ ID#Cellu ratio LPF Cellu ratio LPF Cellu ratio LPF UPN1 Hyper 0.8 4 Hyper0.7 10 Hyper 0.6 13 UPN3 Hyper 0.8 2-3 Hyper 0.3 6 UPN5 Norm 2 2 Norm0.8 6 Norm 0.5 6 UPN7 Hyper 1 1 Hyper 0.3 8 Hyper 0.03 13

There was no decrease in marrow cellularity upon hematopathology review(Table 3, above), which was blinded to treatment status. In UPN3, theinterim bone marrow aspirate was technically unsuccessful.

Non-hematologic toxicity: NCI Toxicity Criteria were used to assesstoxicity. No local toxicity occurred at SQ injection sites. Nonon-hematologic toxicity occurred. No subjects described nausea,vomiting, diarrhea, constipation, or decreased appetite.

Efficacy: total Hb increased from 7.6±2 to 9.6±1.8 (mean±2 SD ofpre-treatment to peak Hb, paired t-test p<0.001). Both the absolutereticulocyte count (ARC) (p=0.0006) and total bilirubin (p=0.01,2-tailed paired t-test) decreased during treatment. The ARC correlatedinversely with tHb (p<0.0001). In SCD, abnormal exposure of moleculessuch as phoshphatidyl-serine on the RBC surface and adhesion of RBC toendothelial cells/endothelial damage can trigger coagulation andinflammatory pathways. RBC adhesion to both TSP and laminin decreasedfollowing cycle 1 (p<0.005). In agreement with previous reports (Tomer,A., et al. 2001 J Lab Clin Med 398-407; Francis, R. B., Jr. 1989Haemostasis 19:105-111; Peters, M., et al. 1994 Thromb Haemost71:169-172), increased levels in markers of active coagulation,Thrombin-antithrombin (TAT), FI+2 and D-dimers, were noted at baseline.Treatment decreased D-dimer levels, a measure of fibrinolysis ofcross-linked fibrin (p<0.04), while markers of thrombin generation, TATand FI+2, decreased, but not to a statistically significant extent. Theadhesion molecule soluble VCAM (s VCAM-I) and von Willebrand factorpeptide (VWFpp) are released from damaged endothelial cells, levels ofboth molecules decreased with treatment (p<0.05). C-reactive protein(CRP), a marker of inflammation, was elevated at baseline and, althoughthere was a downward trend with therapy, it was not statisticallysignificant (p=0.18) (see Table 4, below).

TABLE 4 Changes in surrogate clinical endpoints. Post Post NormalPretherapy Cycle 1 P* Cycle 2 P** Range Measures of Adhesion to 1570 ±170 690 ± 150 <0.001 910 ± 160 <0.001 <60 RBC adnesion TSP to (RBCs/mm²)endothelium Adh. to 3470 ± 500 1950 ± 300  0.004 1570 ± 210  <0.001 <250laminin (RBCs/mm²) Measures of D-Dimef 490 ± 90 320 ± 50  0.02 300 ± 50 0.03 <400 thrombin (ng/mL) generation TAT (ug/L)  7.0 ± 1.7 8.6 ± 2.30.15 5.2 ± 0.9 0.11 1.0-4.1 and F1 + 2  1.75 ± 0.22 1.56 ± 0.16 0.231.41 ± 0.15 0.051 0.04-1.1  fibrirtolysis (nmol/L) Measure of CRP(mg/dL)  1.25 ± 0.27 1.19 ± 0.34 0.80 0.82 ± 0.26 0.18 <0.7 inflammationMeasures of sVCAM 1170 ± 140 930 ± 100 0.01 840 ± 100 0.02 395-714endothelial (ng/mL) cell damage VWFpp (u/dL) 196 ± 26 156 ± 28  0.015144 ± 13  0.049  74-153 Values are mean ± SE; paired 2-tailed t-test. P*= significance of change from pre-therapy to post-cycle 1. P** = changefrom pre-therapy to post-cycle 2.

Example 22. Chronic Decitabine Administration to Seriously Ill SCDPatients (Saunthararajah. Y., et al. 2008 Br J Haematol 141:126-129)

Previous studies of decitabine as a potential disease-modifying agentfor sickle cell disease (SCD) were not designed to demonstrate clinicaleffectiveness. In four SCD patients with severe acute illness on abackground of chronic clinical deterioration over the preceding years ormonths, decitabine (0.1-0.2 mg/kg I-2×/week) was administered off-labelfor periods beyond 12 months. The off-label use of decitabine in SCD wasto provide direct benefit to these patients and not for research.Decitabine was considered because of clinical deterioration andlife-threatening complications despite HU therapy, erythropoietin forrelative reticulocytopenia (hemoglobin <9 g/dl & reticulocytes<250×109/L), decreased availability, and increased transfusion risksfrom >5 red blood cell (RBC) allo-antibodies and auto-antibodies, andineligibility for available protocol therapy.

Hemoglobin increases of >1.5 g/dl occurred within 2-4weeks with maximumhemoglobin increases of 3.5-5 g/dl. Hemoglobin increased through anincrease in reticulocytes and an increase in fetal hemoglobin (theincrease in hemoglobin was not explained by increase in fetal hemoglobinalone). Generally, reticulocyte counts increased during the first 2-8weeks of therapy. Reticulocyte trends reversed after hemoglobinlevels >9 g/dl. This was most obvious in Patient A, who was notreceiving exogenous erythropoietin. Durable symptom and performancestatus improvement during 4-12 months of follow-up contrasted withsevere and deteriorating pre-decitabine trends.

Of note, all patients had severe acute illness on a background ofchronic deterioration and progressive anemia over the preceding years ormonths; the follow-up period ranging from 4-12 months alloweddocumentation of durable clinical improvement that contrasted obviouslywith clinical status and trends in the preceding months; although 3 ofthe 4 patients were on concurrent erythropoietin, it had beenadministered at stable doses for more than 6 months with progressiveanemia and recurrent severe anemia exacerbations; although 2 of the 4patients received transfusions during decitabine therapy, these do notexplain the durable increases in hemoglobin and eventual transfusionindependence. The severe and complicated clinical circumstance in thesepatients is not typically represented in clinicaltrials. Therefore, thisdescription can complement the clinical studies and provide additionalguidance regarding dose, schedule, anticipated toxicities and inclusioncriteria.

Example 23. Pharmacodynamics of Decitabine Alone and in Combination withTHU

Significant inter-individual variability in pharmacodynamicresponses inbaboons. The biologic relevance of differences in pharmacokineticresponses to oral decitabine were examined in a different set ofbaboons, in which large differences in individual pharmacodynamicresponses (fetal hemoglobin expression) and biologic activity(neutrophil counts) were noted (Table 5, below).

TABLE 5 Pharmacodynamic responses following SQ and oral administrationof decitabine to baboons. Dose Pre- Post- ANC Animal Drug Route mg/kg/dHbF % HbF % nadir 6974 Dac SQ 0.52 7.7 68.8 1668 Dac-m Oral 9.35 10.340.5 2000 Dac-m Oral 18.7 7.7 67.8 1659 7001 Dac SQ 0.52 7.2 66.1 1491Dac Oral 9.35 6.6 17.4 1331 Dac-m Oral 4.1 7.4 13.1 1417 7002 Dac SQ0.52 7.4 86.3 524 Dac m Oral 6.7 7.9 31.4 481 Dac-m Oral 9.35 8.3 62.3463 7254 Dac SQ 0.52 8.4 76.3 1320 Dac Oral 18.7 7.1 68.1 1969 7257 DacOral 9.35 6.0 72.0 749

Fetal hemoglobin expression, HbF %, pre and post treatmentwith theindicated dose of decitabine. Decreases in neutrophil counts, anotherexpected biological effect of decitabine, paralleled the increases inHbF %. Oral treatment was with decitabine or a slightly modifieddecitabine (decitabine-mesylate—Dac-m). Inter-individual variability inpharmacodynamic responses to the same dose of oral decitabine arehighlighted by the boxes.

Example 24. The Administration of THU Prior to Decitabine DecreasesInter-Individual Variability in Decitabine

Similarly, administration of oral THU prior to oral decitabine in twoother baboons (PA 6974 and 7001) produced a substantial increase in thepharmacodynamic responses (fetal hemoglobin expression) to oraldecitabine (Table 6, below).

TABLE 6 Pharmacodynamic responses (fetal hemoglobin expression, HbF %)were substantially enhanced by administering oral decitabine after oralTHU. THU Dose oral Pre- Post- Animal Drug Route mg/kg/d 20 mg/kg HbF %HbF % 6974 Dac Oral 9.35 − 10.3 40.5 Dac Oral 0.3 + 3.2 28.4 7001 DacOral 9.35 − 6.6 17.4 Dac Oral 1.42 + 6.2 61.6

Decitabine was administered at the doses indicated in two consecutiveblocks of 5 days each. THU at the indicated doses was administered 60minutes before each decitabine dose.

Example 25. The Effects of Decitabine (DAC) on DNMT1 Depletion, DNADamage, and Apoptosis in Normal Hematopoietic Precursors

FIGS. 19A-19E illustrates effects of DAC on DNMT1 depletion, DNA damage,and apoptosis in normal hematopoietic precursors. (FIG. 19A) DAC 0.005μM depletes DNMT1 in normal hematopoietic precursors. Normal CD34 cellswere isolated from cord blood. DAC 0.005 μM was added once daily on days1-4 and DNMT1 was quantified by Western blot on day 5. (FIGS. 19B-C)DAC >0.5 μM was required to induce measurable DNA damage. Twenty-fourhours after DAC or AraC exposure, DNA damage was measured byflow-cytometric assessment for phosphorylation of histone H2AX (γH2AX;B) or the Fast Micromethod for DNA scission (FIG. 19C). Equimolar levelsof AraC were used as positive controls. Gray histogram is the isotypecontrol. (FIG. 19D) DAC >0.5 μM was required to induce apoptosis.Twenty-four hours after DAC or AraC exposure, apoptosis was measured byflow-cytometric assessment for annexin staining. Doubleannexin/7-aminoactinomycin D (7AAD)—positive cells represent lateapoptosis/necrosis. (FIG. 19E) DAC up to 0.5 μM in combination with THUdid not cause significant DNA damage, as measured by flow-cytometricassessment of H2AX levels 24 hours after addition of the drug to normalhematopoietic precursors. Results are expressed as a percentage ofvehicle treated controls.

The effects of DAC on DNA damage, apoptosis, and DNMT1 levels in normalCD34⁺ hematopoietic precursors isolated from cord blood was examined toidentify a concentration range that depletes DNMT1 without cytotoxicity.AraC, a cytidine analog that terminates DNA chain synthesis, was used asa positive control for DNA damage and apoptosis induction. DAC 0.005 μMalone and in combination with THU 0.010 substantially depleted DNMT1(FIG. 19A). Concentrations of DAC up to 0.50 μM did not cause measurableDNA damage, as measured by levels of phospho-H2AX (γH2AX) and the FastMicromethod for DNA scission (FIG. 19B-C), or apoptosis, as measured byannexin staining (FIG. 19D). DAC at 1 μM caused measurable DNA damageand apoptosis (FIG. 19B-D), although not to the same extent as AraC 0.50μM (FIG. 19B-D). In the presence of THU 0.1 or 100 μM DAC up to 0.50 μMdid not significantly increase DNA damage, as measured by H2AX (FIG.19E).

Example 26. Effects of Different Routes of Administration on the DACConcentration-Time Profile

In vitro studies have suggested that high peak DAC concentrations areunnecessary for DNMT1 depletion and may increase the risk forcytotoxicity. To compare the effect of different routes ofadministration on the DAC concentration-time profile, plasma DAC levelswere compared after IV or SC administration versus oral administrationto the same animals (washout period of ≥2 weeks between drugadministrations to the same animal). FIGS. 20A-20D illustrate plasmaconcentration-time curves following intravenous (IV) decitabine (DAC) 10mg/m2 (0.5 mg/kg), subcutaneous (SC) DAC 10 mg/m2 (0.5 mg/kg) and oralgavage (Oral) DAC 200 mg/m² (10 mg/kg) administration to baboons. Someanimals also received oral DAC 100 mg/m² (5 mg/kg) 60 minutes after THU400 mg/m2 (20 mg/kg). Blood was collected for up to 7 time-points afteradministration and plasma concentrations were determined using LC/MSMS.The data shows that administration by the oral route produces lower peaklevels and a longer half-life than IV or SC administration.

FIG. 20A shows IV and oral administration in PA7472. FIG. 20B shows IVand oral administration in PA7482. FIG. 20C shows SC and oraladministration in PA7254. FIG. 20D shows SC and oral administration inPA7258.

In baboon number PA7472, IV DAC 10 mg/m² produced a peakdrug levelof >1.3 μM (300 ng/mL) and a half-life <5 minutes. In contrast, the peakdrug level with oral DAC 200 mg/m² was <0.015 μM and half-life >100minutes (FIG. 20A). A lower peak drug level but longer half-life withoral compared with IV administration was also observed in baboon numberPA7482 (FIG. 20B). In baboon number PA7254, SC DAC 10 mg/m² produced apeak drug level of 0.36 μM and a half-life of <50 minutes. In contrast,the peak drug level with oral DAC 200 mg/m²was <0.015 μM and thehalf-life was >150 minutes (FIG. 20C). A lower peak drug level but alonger half-life with oral compared with SC administration was alsoobserved in baboon number PA7258 (FIG. 20D).

Example 27. Identification of Dose and Timing of Oral THU to IncreaseOral Bioavailability of DAC in Nonhuman Primates

In 2 female baboons, PA7470 and PA7484 (selected for high and lowbioavailability of oral DAC alone, respectively), THU 400 mg/m² (20mg/kg) 60 minutes before DAC 100 mg/m² (5 mg/kg) produced higher DACconcentrations than THU 40 mg/m² (2 mg/kg; FIG. 21A-B). In these samebaboons after a washout period, THU 400 mg/m² 60 minutes before DACproduced higher DAC concentrations than THU 400 mg/m² administeredsimultaneously or 30 minutes before DAC (FIG. 21A-B).

FIG. 21A: Baboon PA7470 treated with different doses of THU and DAC anddifferent timing between the drugs. THU 400 mg/m2 (20 mg/kg) producedhigher DAC concentrations than THU 40 mg/m2 (2 mg/kg). THU 400 mg/m2 60minutes before DAC produced higher DAC concentrations than simultaneousor 30 minute prior administration of THU. FIG. 21 B: Baboon PA7484treated with different doses of THU and DAC and different timing betweenthe drugs. THU 20 mg/kg produced higher DAC concentrations than THU 2mg/kg. THU 20 mg/kg 60 minutes before DAC produced higher DACconcentrations than simultaneous or 30 minute prior administration ofTHU.

Example 28. Effect of THU on DAC Oral Bioavailability andInterindividual Variability in Nonhuman Primates

Prior administration of oral THU increases oral bioavailability anddecreases interindividual variability in pharmacokinetics of DAC. Eightfemale baboons were treated with oral DAC 200 mg/m² (10 mg/kg). Themedian AUClast with oral DAC alone was 463 min/ng/mL, with a range of190-6279 min/ng/mL, an approximately 33-fold variation, and acoefficient of variation of 1.41 (Table 7 and FIG. 22A).

TABLE 7 AUClast after oral DAC versus oral THU-DAC in the same baboonsAUC_(last) (min*ng/mL)^(#) Decitabine Decitabine THU 20 mg/kg 60 Weight10 mg/kg 5 mg/kg mins before Baboon Number (kg) alone alone Decitabine 5mg/kg PA7482 12.3 49.98 Not done Not quantifiable PA7484 11.5 190.35252.73 5621.05 PA7256 19.8 299.02 Not done 760.27 PA7472 10.7 327.25 Notdone 444.825 PA7254 14.4 463 Not done 2587 PA7255 19.9 807.8 Not done533.47 PA7258 12.6 2863.6 Not done 2284.48 PA7470 9.6 6278.78 1219.987515.32 Mean ± SD 1604 ± 2262 2821 ± 2749 Median ± IQR  463 ± 2565 2284± 5088 Median ± IQR per 160 ± 226  457 ± 1017 mg of decitabine*Fold-variation ~30-fold ~14-fold Coefficient of 141 97 Variation^(#)AUC_(last) calculated over 240 minutes for decitabine alone, 180minutes for THU-decitabine. *p = 0.02. Wilcoxon test. SD = standarddeviation. IQR = inter-quartile range

After a washout period of >2 weeks, the same animals were treated withDAC 100 mg/m² (5 mg/kg; half the dose used for the DAC aloneexperiments) 60 minutes after oral THU 400 mg/m². The median AUC_(last)with THUDAC was 2284 min/ng/mL, with a range of 534-7515 min/ng/mL, anapproximately 14-fold variation, and a coefficient of variation of 0.97(Table 7 and FIG. 22B). The average DAC Cmax was 0.05 μM (10.85 ng/mL)for DAC alone and 0.12 μM (26.98 ng/mL) for THU-DAC (DAC at half thedose; FIG. 22A-B). The decrease in the coefficient of variation in theTHU-DAC group was because the largest AUC_(last) increases occurred inanimals that had the poorest bioavailability with DAC alone (Table 1 andFIG. 22C). The AUC_(last) difference between DAC alone and THU-DAC wasstatistically significant (P=0.02 by Wilcoxon test; Table 7), eventhough AUC_(last) was calculated at 25% more time for DAC alone (240minutes vs 180 minutes for THU-DAC). The last measured DAC plasma levelwas the highest level observed in 4 of 7 THUDAC-treated animals with DAClevels measurable at more than one time point, and half-life was notreached at 180 minutes in any of these 7 animals (FIG. 22B). Therefore,AUC_(last) values for THU-DAC are likely to be underestimates.

FIG. 22A illustrates DAC concentration-time profiles in 8 baboonsadministered oral DAC 200 mg/m² (10 mg/kg). FIG. 22B illustrates DACconcentration-time profiles in the same 8 baboons administered DAC athalf the dose (100 mg/m² [5 mg/kg]) 60 minutes after THU 400 mg/m² (20mg/kg; THU-DAC). PK measurements went to 180 instead of 240 minutesbecause of the allowable duration of anesthesia. The largest increasesin AUC_(last) with coadministration of THU were seen in animals withlower intrinsic oral bioavailability of DAC (FIG. 22C). Histograms showthe distribution of AUC_(last) in 7 animals administered DAC aloneversus the same 7 animals receiving DAC at half the dose after THU. AUCmeasurements went to 180 instead of 240 minutes in the THU-DAC groupbecause of the allowable duration of anesthesia.

Example 29. Effect of THU on Oral DAC Pharmacokinetics in Mice

To more completely evaluate the effect of THU on DAC pharmacokinetics toan extent not possible in nonhuman primate studies, studies wereconducted in mice. Using oral gavage, female CD-1 mice were administeredTHU 400 mg/m² (167 mg/kg) 60 minutes before DAC 0.3, 0.6, or 1.2 mg/m²(0.1, 0.2, or 0.4 mg/kg, respectively) or DAC 1.2 mg/m² alone (vehiclewas administered instead of THU) twice a week for 3 weeks, andpharmacokinetics were measured after administration of the last dose(day 16). FIG. 23 illustrates the DAC concentration-time profile in miceadministered DAC alone or DAC 60 minutes after THU. Drugs wereadministered by oral gavage. Dots show values from 3 mice for each timepoint in each treatment group. THU-DAC indicates DAC 0.4 mg/kg 60minutes after THU 167 mg/kg. DAC indicates DAC 0.4 mg/kg 60 minutesafter vehicle.

THU extended the period of DAC absorption: the concentration-time curvewas widened by early and late absorption (2-parallel first-orderabsorption; FIG. 23). This effect of THU on the shape of the DACconcentration-time profile was reflected in a 9-fold increase inAUC_(total) (from 8.45 min/μM with DAC alone to 76.24 min/μM withTHU-DAC), compared with a 2.5-fold increase in C_(max) (from 0.251 to0.617 μM; Table 8). There was a linear relationship between oral THU-DACdose and DAC C_(max) and AUC_(total) (Table 8). The coefficients ofvariation for AUCtotal were substantially lower than in baboons: 0.24for DAC 1.2 mg/m² alone and 0.15 for THU-DAC 1.2 mg/m² (Table 8).

TABLE 8 Pharmacokinetic parameters after oral DAC versus oral THU-DAC infemale mice (mean values from 3 mice at each time point in each group).Dec THU-Dec THU-Dec THU-Dec 0.4 Group 0.1 0.2 0.4 only Decitabine dose,mg/kg 0.1 0.2 0.4 0.4 THU dose, mg/kg 167 167 167 0 adminstered 60 minsbefore decitabine) AUCt (min*μM) 1790 35.64 76.24 345 Cmax (μM) 0.1380.338 0.617 0.251

Example 30. Pharmacodynamic Effects in Nonhuman Primates of Repeat-DoseOral THU-DAC

To evaluate pharmacodynamic effects with repeat-dose administration,oral THU-DAC 2×/wk for 8 weeks was administered to 4 baboons. Twoanimals, one each with relatively low and high oral THU-DACbioavailability (baboon numbers PA7472 and PA7470, respectively) per thepharmacokinetic studies (Table 7), received oral DAC 5 mg/m² after oralTHU 400 mg/m². Similarly, a pair of animals from the low and high end ofthe oral THU-DAC pharmacokinetic range (baboon numbers PA7482 andPA7484, respectively) received oral DAC 10 mg/m² after oral THU.

FIGS. 24A-24E and FIGS. 25A-25F illustrate pharmacodynamic effects ofrepeatdose oral THU-decitabine in non-human primates. In FIGS. 24A-24E,a baboon with relatively low and a baboon with relatively high oralTHUDAC bioavailability in the pharmacokinetic (PK) studies (baboonnumbers PA7472 and PA7470, respectively) received DAC 5 mg/m², andanother pair from each end of the PK range (baboon numbers PA7482 andPA7484, respectively) received DAC 10 mg/m². DAC was administered 60minutes after THU 400 mg/m²2×/wk for 8 weeks. FIG. 24A shows plateletcounts during drug administration. FIG. 24B shows absolute neutrophilcounts during drug administration. FIG. 24C shows phospho-H2AX (γH2AX)labeling of BM cells 96 hours after THU- DAC administration in week 8 inbaboon number PA7472. Positive control HeLa cells treated withcamptothecin 10 μM. Negative control vehicle treated HeLa cells. FIG.24D shows HbF expression during treatment. FIG. 24E shows decrease inmethylation of developmentally responsive CpG in the γ-globin gene (HBG)promoter after drug administration in baboon numbers PA7472 and PA7484.Based on the human β-globin gene locus(http://www.ncbi.nlm.nih.gov/nuccore/U01317), the coordinates of theseCpGs were 33105, 33221, 34425, and 34483. Mass spectrometry was used tomeasure methylation levels in DNA extracted from erythroid precursorsisolated from fetal BM (FBM), adult BM (ABM), before THU-DAC(pre-THU-DAC), and after 8 weeks of 2×/wk oral THU-DAC (post-THU-DAC) inbaboon numbers PA7472 and PA7478, and from WBCs. Box-plot boundaries:interquartile range; horizontal line, median; +, mean; small boxes,outlying values; whiskers, range of values. P values are by Wilcoxontest.

In FIGS. 25A-25F, decitabine 5 mg/m2 (PA7484) or 2.5 mg/m2 (PA7472) 60minutes after THU 400 mg/m2 3×/week was administered for 5 weeks toPA7484 and for 3 weeks to PA7472. First dose day 1, last day oftreatment day 31 (PA7484) or day 19 (PA7484). FIG. 24A shows plateletcounts in PA7484. FIG. 24B shows absolute neutrophil counts (ANC) inPA7484. FIG. 24C shows HbF % in PA7484. FIG. 24D shows platelet countsin PA7472. FIG. 24E shows ANC in PA7472. FIG. 24F shows HbF % in PA7472.

Oral DAC 5 and 10 mg/m² after THU was expected to produce a Cm_(max) ofapproximately 0.006 and 0.012 μM, respectively, because there is alinear relationship between THU-DAC dose and pharmacokinetic parameters(FIGS. 21A-21B and FIGS. 25A-25F), and oral DAC 100 mg/m² after THUproduced an average Cmax of 0.12 μM. Noncytotoxic modification ofhematopoietic differentiation by DAC is expected to produce increases inplatelets and decreases in neutrophil counts, as suggested by previousin vitro and clinical studies. In contrast, cytotoxic therapy isexpected to produce concurrent decreases in platelets and neutrophils.In the 4 baboons, platelet counts increased during weeks 1-4 of drugadministration (Table 9 and FIG. 24A). Although this upward trendreversed in 2 baboons during weeks 6-8 of drug administration, plateletcounts did not decrease below the lower limit of normal (FIG. 24A).Neutrophil counts decreased during weeks 1-3 of drug administration(Table 9 and FIG. 24B), but then increased again or remained stableduring weeks 4-8 of drug administration (FIG. 24B). In baboon numberPA7472, a BM aspirate 96 hours after THU-DAC administration providedsufficient cells for analysis of DNA damage/repair by γH2AX. There was asmall increase in γH2AX compared with negative control that was notsuggestive of major DNA damage, although early DNA damage would havebeen missed (FIG. 24C).

TABLE 9 Repeat-dose administration of oral THU-DAC in 4 baboons: effectson HbF percentage and neutrophil and platelet counts DAC Peak ANCPlatelet count Baboon dose, Pretreatment HbF, nadir, maximum, no. mg/m²⁺Schedule HbF, % % ΔHbF ×10⁹/L ×10⁹/L 7470 5 2×/wk for 8 wks 9.9 29.329.3 2.13 895 7472 5 2×/wk for 8 wks 12.8 30.1 30.1 1.48 600 7482 103×/wk for 3 wks 3.5 23.8 20.3 2.58 1004 2×/wk for 5 wks 7484 10 2×/wkfor 8 wks 4.2 23.1 23.1 1.68 1029 Two of these animals had relativeilylow bioavailability (baboons PA7472 and PA7482) and 2 had relativelyhigh oral THU-DAC bioavailability (baboons PA7484 and PA7470) aocordingto the pharmacokinetic studies. ANC indicates absolute neutrophil count.*Adininistered after THU 400 mg/m².

In additional experiments, oral THU-DAC was administered 3×/wk with 50%of the various DAC doses, again with concurrent increases in plateletand decreases in neutrophil counts (FIGS. 25A-25F).

One potential application of long-term DNMT1-depleting therapy is toincrease HbF (α2γ2) expression to treat hemoglobinopathies such assickle cell disease and β-thalassemia. In all 4 animals, HbF levelsincreased progressively during weeks 1-4 of drug administration (Table 9and FIG. 24D). From weeks 5-8, there was a small decrease from thesepeak HbF levels in the 2 animals receiving the higher dose of DAC (10mg/m2), whereas levels continued to increase in the 2 animals receivingDAC 5 mg/m2 (FIG. 24D), producing higher peak HbF levels (Table 9).Progressive HbF increases were also noted with 50% of the DAC dosesadministered 3×/wk after THU (FIGS. 25A-25F). One intended moleculareffect of therapy is to decrease methylation at promoter CpGs thatregulate the expression of target genes (e.g., the γ-globin gene [HBG]promoter CpG). Methylation levels of 4 CpG sites in the HBG promoter wasmeasured by mass spectrometric analysis of DNA extracted from BMerythroid precursors (from baboon numbers PA7472 and PA7484). Aftertreatment with oral THU-DAC, the methylation levels of these HBGpromoter CpGs decreased by approximately 20% compared with pretreatmentmethylation levels (P=0.007; FIG. 24E). The relevance of these CpG sitesto physiologic regulation of HBG expression was suggested by significanthypomethylation in DNA isolated from fetal BM erythroid precursorsversus adult BM erythroid precursors and by significant hypermethylationin DNA isolated from peripheral WBCs (FIG. 24E).

In both baboons and mice, oral administration of THU to inhibit CDAbefore oral administration of DAC extended DAC absorption time andwidened the DAC concentration-time profile, as reflected in mice by a9-fold increase in DAC AUC_(total) compared with a 2.5-fold increase inDAC C_(max). Because DNMT1 depletion by DAC can occur at very low druglevels but depends on exposure timing, the wider concentration-timeprofile achieved with oral THU-DAC could have efficacy advantages withregard to DNMT1 depletion without the high peak DAC levels that cancause DNA damage and cytotoxicity.

The baboon model has been accurate and useful in predicting by bodysurface area scaling a safe human equivalent dose for SC DAC treatmentand for combination oral THU and 5-azacytidine therapy. Therefore, theTHU dose (400 mg/m²) and timing (60 minutes before DAC) that are likelyto be useful for human translation were identified by studies inbaboons. In the pharmacokinetic studies in baboons, AUC_(last) estimatesfor DAC alone were calculated over 240 minutes, whereas estimates forTHU-DAC were calculated over 180 minutes (the permissible total durationof anesthesia in the nonhuman primate studies was 240 minutes;therefore, the administration of DAC 60 minutes after THU decreased theduration of sampling in THU-DAC-administered animals). Furthermore, theconcentration-time profiles suggested that DAC levels may have continuedto increase beyond the last sampling time in many THU-DAC-treatedanimals. Therefore, the presented values underestimate the increase inDAC bioavailability produced by preceding THU administration in baboons.

Although the murine studies enabled more comprehensive analyses of theeffects of THU on DAC pharmacokinetics, dose-exposure extrapolation bybody surface area scaling from mice to humans is not useful, becausedose for dose, there is a more than 100-fold greater DAC exposure inrodents versus humans. Similarly, there is more than 100-fold greaterexposure of the cytidine analog AraC in mice versus monkeys dose fordose. The reasons for this log-scale increase in cytidine analogexposure in rodents compared with primates are unknown.

An important limitation of DAC and other cytidine analogs has been theinter-individual variations in pharmacokinetics, toxicity, and efficacythat are associated with single nucleotide polymorphisms in CDA. THU, byinhibiting CDA, may attenuate the role of these pharmacogeneticvariations in cytidine analog pharmacokinetics and pharmacodynamics. Forexample, in baboons, THU decreased substantial interindividualvariability in DAC pharmacokinetics compared with oral DAC alone.Similarly, THU decreased substantial interindividual variability in HbFelevations (peak levels ranged between 23% and 30%) compared withprevious experience with oral DAC alone (peak levels ranged from10%-60%). However, the basis for interindividual variability in baboonshas not been characterized. Consistent with a genetic contribution tointerindividual variability, variability in mice of the same gender wassubstantially less than in baboons.

Combination therapy with THU may offer other advantages, because CDAup-regulation is a putative mechanism of cancer cell resistance tocytidine analogs, and cancer cells may find sanctuary from cytidineanalogs in tissues expressing high levels of CDA.

One potential clinical application of DNMT1-targeted therapy is toincrease HbF expression as a treatment for sickle cell disease andβ-thalassemia. In baboons, repeat administration of oral THU-DAC using aDAC dose that would produce peak DAC concentrations less than 0.2 μM wasnot myelotoxic, hypomethylated HBG promoter CpG, and produced largecumulative increases in HbF expression in RBCs.

For the purposes of targeting DNMT1, the RNA incorporation that occurswith 5-azacytidine is an off-target effect. Although the present dataindicate that noncytotoxic DNMT1 depletion with DAC is possible, DNMT1depletion occurs after DNA incorporation of DAC and postreplicativeimmobilization of DNMT1, and therefore genotoxicity remains a possibleside effect that will have to be evaluated in clinical studies with oralTHU-DAC. In conclusion, in baboons and mice, preceding administration oforal THU substantially increases oral bioavailability of DAC, creates aconcentration-time profile that suits the purpose of DNMT1 depletionwith less cytotoxicity, and decreases interindividual variability.Therefore, combination oral THU- DAC may facilitate more accessible,safe, and efficacious DNMT1-targeted therapy.

Surprisingly, while the baboons receiving the 10 mg/m² dose of DACshowed a decrease from peak HbF levels in weeks 5-8, HbF levelscontinued to increase in the baboons receiving DAC 5 mg/m² (FIG. 24D),resulting in higher peak HbF levels (see Table 9). Progressive HbFincreases were also noted with 50% of the DAC doses administered 3×/wkafter THU (FIGS. 25A-25F).

Although certain embodiments have been illustrated and described herein,it will be appreciated by those of ordinary skill in the art that a widevariety of alternate and/or equivalent embodiments or implementationscalculated to achieve the same purposes may be substituted for theembodiments shown and described without departing from the scope. Thosewith skill in the art will readily appreciate that embodiments may beimplemented in a very wide variety of ways. This application is intendedto cover any adaptations or variations of the embodiments discussedherein. Therefore, it is manifestly intended that embodiments be limitedonly by the claims and the equivalents thereof.

1-20. (canceled)
 21. A composition for oral administration comprising atherapeutically effective amount of decitabine and tetrahydrouridine anda pharmaceutically acceptable excipient, wherein the tetrahydrouridineis bio-available about 15 to about 180 minutes before the decitabine.22. The composition of claim 21, wherein the composition is in a soliddosage form.
 23. The composition of claim 21, wherein the composition isin form of a capsule.
 24. The composition of claim 23, wherein thedecitabine is located within the capsule.
 25. The method of claim 21,wherein decitabine is coated with a coating.
 26. The composition ofclaim 21, wherein the composition is effective to produce a decitabineplasma concentration of 0.005 μM to 0.05 μM in a human subject for atleast 60 minutes, wherein the decitabine plasma concentration does notexceed 0.2 μM.
 27. The composition of claim 21, wherein the compositioncomprises decitabine and tetrahydrouridine in amounts effective to causea decitabine plasma concentration of between 0.005 μM and 0.1 μM in ahuman subject for at least 60 minute.
 28. The composition of claim 21,wherein the decitabine is present in an amount effective for treating ablood disorder in a subject.
 29. A method for treating a hematologicalor solid malignancy in a subject, the method comprising administering tothe subject the composition of claim
 21. 30. A method for decreasing theinter-individual variation in decitabine pharmacokinetics and/orclinical effects in subjects, comprising administering to the subjectsthe composition of claim
 21. 31. A composition for oral administrationcomprising about 10 to about 150 mg/m decitabine and about 100 to about500 mg/m tetrahydrouridine and a pharmaceutically acceptable excipient.32. The composition of claim 31, wherein the composition is in a soliddosage form.
 33. The composition of claim 31, wherein the composition isin form of a capsule.
 34. The composition of claim 33, wherein thedecitabine is located within the capsule.
 35. The method of claim 31,wherein decitabine is coated with a coating.
 36. The composition ofclaim 31, wherein the composition is effective to produce a decitabineplasma concentration of 0.005 μM to 0.05 μM in a human subject for atleast 60 minutes, wherein the decitabine plasma concentration does notexceed 0.2 μM.
 37. The composition of claim 31, wherein the compositioncomprises decitabine and tetrahydrouridine in amounts effective to causea decitabine plasma concentration of between 0.005 μM and 0.1 μM in ahuman subject for at least 60 minute.
 38. The composition of claim 31,wherein the decitabine is present in an amount effective for treating ablood disorder in a subject.
 39. A method for treating a hematologicalor solid malignancy in a subject, the method comprising administering tothe subject the composition of claim
 31. 40. A method for decreasing theinter-individual variation in decitabine pharmacokinetics and/orclinical effects in subjects, comprising administering to the subjectsthe composition of claim
 31. Attorney's Docket No.: 121946-251423DB061-USC3 Application No.: 16/685,956